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| Q&A
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I didn't attach the gel exclusion data sheets to my lab report because they aren't the typical signed data sheets that we must include with all of our reports. Plus, the data is the same for all of my section and the information is stored in the gel exclusion database. Will I have points taken off for this? If so, can I still turn them in?
Hi Z,
It should be okay, but I will have to confer with the TA about this.
Hi Dr. Kim. Where can we pick up our labs and midterms? thanks. Brad
Hi Brad,
Most likely the reports will be available next Thursday. I will announce times. Unfortunately, I will not have anyone around to handout graded reports and midterms. But I will be in next Thursday. If you are away from campus by, then you can pick up the reports next quarter.
Hi Dr. K when we draw a plot log Mr vs. distance, should we use logarithmic scale or just plane scale?
You should use a plane scale. If you plotted Mr vs. distance and wanted a linear relationship, you would use a log scale.
Hi, Prof.Kim, a few questions about SDS-page lab report. #1) For question number 2, it says we have to mark our reference protein. There are five bands on my first lane. So, those bands will be on my first lane, right? 2#) For question number 3, i thought we only had 4 reference proteins (GPDH, BSA, Trypsin Inhib, and Carbonic Anhydrase, but then why there are 5 rows? Am I missing something? Thank you for answering my questions.
I hope you are using the course reader report instructions for I do not see where you are getting this information. You should know the order in which your samples were loaded. And know the order will dictate which lane you should use. Not just the first lane.
As for the 5 rows, just fill in the first four.
Professor, For the Michaelis-Menten plot of our Lab 6 LDH Kinetics data, is the only way we can calculate Vmax and Km purely observational (i.e. interpolating the maximum V0 when [Substrate]=1.0) or is there another way we can calculate these values? Should we just use the Km and Vmax values we determined from the double-reciprocal plot and compare them to the Michalis-Menten plot and either say they are different/similar for Question 3B of the Kinetics lab report?
For the MM plot, I expect you to make a "by eye" observation of the Vmax and Km. Make your best estimate. You should find they are still different to the LB plot.
Hi Dr.Kim, for kinetic report,question #1, it's asking fraction [protein] and [enzyme], does it refer to the diluted amount or undiluted amount? thank you.
You should enter the undiluted amount. And then, when you determine your kcat value, you will account for the dilutions in order to obtain the enzyme concentration in reaction mixtures.
Hi Dr Kim. Just to make sure. when is the kinetics lab due? (for thurs lab)
That would be Tuesday (today)
Hi Dr. Kim, when should we expect our final grade, and when can we pick up all of out Post Labs and Exam 5B? Thank you
The final grades will completed by next week, hopefully by 21, although the final grade deadline is the 23. As for picking up reports, I will announce when the reports can be picked up after I meet with the TAs tomorrow.
Exam 5b can be picked up next week by Monday. I will also announce exact times when you can pick up the exam and regraded material.
hi dr. kim, i am in lab 1I and our gel data was to be ready for pick up today. i went by twice already and it is not there but i saw data for other lab sections. i am wondering where it is and when it will be ready before i make another trip to campus. thanks!
Hi Katie,
The data is in 1060 now. If you need the data, and the room is locked, I will put it outside of 1060 for you to pick up.
Are there any office hours finals week?
Hi B,
Unfortunately, there will be no office hours. But if you need assistance, please e-mail me directly.
Hi Dr. Kim, When I analyzed the SDS-PAGE and gel exclusion data, I got 5 subunits for LDH, probably because the LDH and GDPH bands ran together and couldn't be accurately measured. The postlab instructions for the kinetics lab say to use the number of subunits obtained in the gel lab to do the calculations. Should I use that number or the correct one, 4?
Hi Jane,
Just use the subunit molecular weight. Not the number of subunits.
Hi, I'm stil relaly confused how to calculate Kcat, the turnover number. Could you please help me? Also, I thought I heard in class that the kinetic lab was due on monday, the 12, is that true?
Hi Henry,
If your section is on Wednesday, then Kinetics report is due the 12th. As for Kcat, remember that Vmax = kcat [E]total. You can determine Vmax from your data, and you can calculate the [E]total. But to determine the [E]total, you need to convert that known mass concentration to molar concentration. You have determined all these values from your other experiments. Think about these values and if you get stuck, get back to me.
Hi Dr. Kim, For the kinetics report, can we use excel to construct the Michaelis-Menten and double reciprocal plots and then manually draw the best-fit lines? Or should we plot everything by hand? Thanks so much! Christine
Hi Christine,
Sure. That's fine.
i'm a little confused on the sds-page lab. 1. how do we know which marker is which by comparing the lanes on our gel if some of our lanes look identical? 2. the reference point would be the appearance of the first band in each lane right? 3. when measuring distance traveled,is this the distance from the top of the gel to the appearance of the first band?
Hello,
1) Yes...compare lanes for identification. Its a bit tricky since you will see many of the standards might have some background bands that need to be ignored, and even your unknown sample may have some too as well.
2&3) The reference point for measuring distance can be the top of the resolving gel. If you wanted a reference distances, in case you were comparing separate lanes (which you are not for our lab), then you would need either a protein band or dye that you would have in each lane for a reference distance as shown in the handout I gave in lecture.
Dr. Kim, after we have plotted our double reciprocol plots and have our y-int (absorbance value). How do we go from this value to mM/min. I know we divide by the molar absorptivity (do we use 6220 or the mol. abs. of pyruvate?) After we put it into units/ml then what? Is that the final value?
Hi G,
Your assay looks at NADH. So converting the value of A/min to mM/min is the conversion to get how much NADH concentration is converted per minute. Keep in mind that pyruvate should have a 1:1 stoichiometry so the final value will be equivalent to the amount of pyruvate converted. And the final value is the rate activity concentration in the reaction tube, not the undiluted stock's concentration. That undiluted value is not even necessary for the evaluation. We want to know the differences in rate activity concentration in the reaction mixtures for the varying substrate concentrations. So the Vmax should be handled in the same way.
Hi professor, I'm confused about the due date of the gel exclusion / sds page lab report.. for section 1k? You mentioned 12th or 14th...
Hi Jae,
For Section K, the official date to have the data available to you is the 12th. And the due date is the 14th. But the data is probably already ready for you to work on the report and turn it in earlier.
Heeey Dr. Kim! When measuring the concentration of active sites of an enzyme, how can we be certain that 1 subunit = 1 active site? Is it possible that 2 subunits might = 1 active site, and how is the number of active sites determined when the enzyme contains subunits of varying molecular weight. Just curious. Thanks!
Good question. It is possible that more than one subunit makes up an active site such as ATP synthase. But for LDH, we assume we already know that one subunit has one active site.
in winter 2005 exam question 1, i was wondering why the two lines (inhibitor and no inhibitor) met at a point not on the y-axis. the answers show that you used 2 different y values to solve for Vmax. i thought for competitive competition, Vmax is the same.
Theoretically, Vmax is the same. But the experimentally, the values are close, but not necessarily identical.
Where do we obtain the data from our gel labs?
In 1060. You can try to pick it up earlier this week, or get it in your lab this week.
for exam 5, #1 in the lab manual, I was wondering how a difference in specific activity by 100x leads to a difference in enzyme concentration by 100x. If they give you amount of protein present, but not [protein], can you still calculate [enzyme] on that?
So the "friend" who used a crude sample would overestimate the total protein amount since there are contaminants. Keep in mind that the molar enzyme concentration can only be determined from the inverse of the MW of the enzyme and the total protein. mole/g x grams of total protein. Also keep in mind that the enzyme activity would be similar since you and the friend would have to dilute the crude or affinity column step to the same enzyme activity concentration for the kinetic experiment. So activity concentration being the same, it should be apparent that the only difference is total protein. For every activity amount in the affinity column assays, there is equal activity in the crude kinetic assays, but 100x more protein due to contaminants.
Oops, disregard the previous question, the "substrate concentrations are different this time"
lol...yup. :)
Prof. Kim, for the upcoming kinetics lab, it says that we will be setting up five test dilutions to find the appropriate dilution, but I can't quite remember if you said we would do these test dilutions or if we should use a theoretical calcuation from the rate data we already have...?
You will use the survey dilutions for this lab. Especially since the substrates are of different concentrations.
hi prof. i just wanted to check when the due date for the gel lab was due. i know it says dec. 5th on the syllabus but for some reason i remember you saying the 12th. thanks
Yes...the due dates are different now. Since the data cannot be available for all labs, we changed the due dates. So for Tuesdays lab, the data of the gels and column will be available on Dec. 8th, and report will be due dec. 12.
Dr.Kim, this is about protocol item on pg 43 lab manual. For column LDH, this refers to peak fractions of elute right? But what is column LDH (2X) referring to? Thank you
Hi Dee,
2x is referring to twice the amount of LDH. So instead of 2.5 µg, you will be adding 5 µg. Notice the adjustment to the amount of water added to that sample. This to ensure that you will be able to visually see the band in case your protein assay values are in error.
Professor Kim, When you ask about what the purified LDH will be used for, do you mean what it will use for in our coming lab? or in clinic or medical world?
Just for the upcoming labs. Unless you are using the LDH for unrelated purposed without my knowledge? :)
Instead of putting an "NA" where data are not available, can I just shade the boxes?
Sure...that looks more aesthetically pleasing.
Dear Dr. Kim, When calculating the deltas for total mg prot and total units enz on the final purification table, do we simply add the delta values from our peak fractions, or are the deltas to be calculated by summing the percent error for each peak fraction? ie, for 0.96+/-0.02 mg and 5.5+/-0.1 mg and 0.99+/-0.01 mg (from our raw data of fractions 9-11), would the final delta for the purification table be 0.02+0.1+0.01=0.13, or (0.02/0.96 + 0.1/5.5 + 0.01/0.99)x(7.5)=0.3?
For adding values of identical units, you can simply add the deltas directly. There is not need to determine % error and then add.
I'm lost. Where does it say in the instructions for the LDH final report to calculate the amount of protein and enzyme per gram of crude extract?
Read the discussion section's instructions in the course reader (page 126).
Professor Kim, Do i need to multiply both the purification factor and % yield by 2 or just the % yield by 2 because I was using only 1 pellet in the affinity column?
Think about the conceptual aspect of this assumption. If you used the other pellet, then you would collect twice the amount of protein and twice the amount of enzyme. That would double your total protein and total enzyme values. That is the only set of values that need to be doubled. Your yield will be automatically doubled. But note, the rate of enzyme to protein do not change. So will the purification factor be doubled?
For the LDH report, does the sample calculations have to be typed?
No...they could be hand written.
Prof.Kim, following up the question about units and mg of pure LDH per 1 g of crude, I'm sorry I think I asked the wrong question. What I meant was would you give hints on how to determine whether our expected pure LDH value is reliable or not. Thank you.
Ah...hints on this. If you know that some of your fractions may contain impurities or proteins that have no LDH activity, then can you make the assumption that the final total protein and units of activity is accurate? If it's not accurate, then how would this impact the amount of LDH per gram of crude protein.
What does "conceptual understanding of values used to monitor the efficiency of the purification step" in the crude result part mean? What kind of things do you expect to write?
What values do you use for monitoring efficiency? Yield and purity. Yield is a pretty straight forward concept, so for a brief example, you would explain that you will compare total activity of one step to the crude's total activity which you will give you your percent yield and indicate how much is lost. As for purification, you would need to explain how comparing values of specific activity demonstrates the increase in purity or purification factor.
Professor Kim, When the report asks to explain the rationale of both assays and what data they will provide in the crude extract part, does it mean we need to state the slope of crude extract in the enzyme assay, and what amount of enzymes it represents after calculation. And what mg/aliquot of proteins it shows in the data, and the amount of the total proteins in the crude after calculation, am I right? Besides, when we report the result, do we need to include the error, like average+/- delta? Thanks a lot!
You can just write out the equation you will use to calculate the final total enzyme, or the steps on the figure itself. In the text, you can refer to the set of calculations in that figure. You need not show average or delta calculations.
sorry another question for the LDH paper... can i include the explanation of the protein assay and enzyme assay and their rationale in the introduction section? or does it have to be in the results section? This eats up much space in the results section. Thanks
Keep your explanation of the assays minimal, but it should be in the result section. You need not write about the extensive details of the assay. For example: In order to determine the concentraiton of protein in each step, we carried out a coomassie blue protein-dye binding assay. Then describe how your carried the ug/aliquot concentration through to the total protein amount. Hope that helps.
For the LDH paper, the direction asks us to provide sample calculations to exemplify how we got the values in the purification table using crude extract data. How are we supposed to use crude extract data if we are trying to show the calculations for yield % and purification factor? Didn't we assume that to be 100% and 1 for crude extract?
Hi Jae,
Example: if you divide the crude total enzyme/crude total enzyme, isn't that the sample calculation. The best way to write this out is step's total enzyme/crude total enzyme x 100% = yield, and then show the calculation.
DO WE NEED TO INCLUDE ALL DATA PRINTOUTS FROM THE FIRST TWO LDH LABS? OR JUST THE ACTUAL TEXT OF THE PROGRESS REPORT WITH THE RAW DATA TABLES?
YOU NEED TO INCLUDE THE ENTIRE PROGRESS REPORT WITH ALL DATA. If you are using the crude data as your figure 1, it is fine that you pull the data sheet from the progress report and place it into the main body of your paper.
Prof. Kim, for determining the units and mg of pure LDH from 1 g of crude, can you give hints on how to decide whether we have successfully purify the LDH or not. Thank you.
The value of units and mg of LDH from 1 g of crude is not directly related to the success of obtaining pure enzyme. Pure LDH can only be derived from the elution profile's elute fraction values. You can compare a certain value of your elute fractions and that will indicate the success of purity. The only other area in which you can determine the success of purification is from other published data sources, but that is optional for this report.
By g of crude extract, do you mean the g of meat that we started out with? If not, then how do we convert the mL of crude to g if density is not known. Thank you.
Hi Gina,
As for the g of crude, that is 1 g of protein isolated at the crude step...how much units or grams of LDH will it yield from our procedure. I still do not know what you are talking about when you refer to mL of crude.
When it says to tell how much LDH can be expected from a gram of crude extract, how do we convert the mL of crude extract to grams? Do we know the density or do you mean the grams of meat that we weighed out initially?
mL to grams? I'm not sure what you mean. Please clarify.
can you explain the interaction between a stacking gel buffer and the running buffer? Will they always be made at the same pH or can they be made at different pHs, and if so, which buffer influences the number of negatively charged glycines? If the pH of the running buffer we will use in lab is ~8.5 then isn't there already a relatively large proportion of negatively charged glycines?
I have attempted to avoid this issue since it would generally confuse many of you. But in the microenvironment between the Cl- layer and the glycine running buffer solution, the concentration of protons in the sample buffer and stacking gel diffuse in the running buffer in that location. So the pH in this microenvironment is basically same as the sample buffer and stacking gel. Thus, glycine remains protonated. Now as the proteins reach the resovling gel, the ion deficient zone will by pass the proteins, and the proteins will soon be in running buffer where the pH is around 8.5. The pH 0f 8.5 is very important in the running buffer so that the there are negatively charged ions (gly-) needed to allow for a current to be adequately passed from the cathode to the where the protein samples are and throughout the rest of the gel. Without these ions, the voltage potential will increase drastical (due to the increase in resistance), and the gel will overheat and in fact cause a crack in either the gel, or the glass plates. This of course is far more complicated for this class and that's why answered this after the exam so you wouldn't have to spend too much time mulling over it.
hi dr kim, if the need to explain results in detail, due to inconsistencies and errors and such, is it ok to go over the limit for the discussion section of the report?
Yes, if there is a significant problem with your data that we would not normally expect for most projects. (basically nonideal results).
Do we need to include error for Yield and Purification Factor?
If you want to have a more comprehensive analysis of your data, you should.
Hi Professor, for calculating enzyme concentration for the fractions, do we multiple my the volume collected for each? And are we only using the 2 highest enzyme and protein peaks for our purification table? Thanks!
Use all elute fractions containing enzyme for total amounts of protein and enzyme, and you need to mulitply the volume for each respective fraction to obtian the total.
I noticed that the last friday lab according to the fall calender is friday dec. 16th. However the report due-date for the last friday lab is dec. 14th. Do we have lab on finals week?
There is no lab during finals week. But reports are still due...your TA will go over that in section.
Hi Dr Kim, for the results section, should I briefly outline how each of the crude, 40%, 60%, and column fractions were prepared and discuss the results of each? I wrote the part for the crude extract only so far and it already comes out to almost a page. Maybe Im not completely understanding how the result section should be written, but could you please guide me as to what i should do different, if any, to stay within the 1 1/2 page limit? Thanks.
Hi Dee,
You probably gave too much basic information regarding the background of crude prep and monitoring assays. Write the paper as if you are writing to someone who has the basic understanding of these assays. So a breif description of the purpose or rationale is just required. For example, no need to describe how the coomassie blue binds to proteins or the three different forms. Besides, your understanding of how these assays and purification steps work will be apparent in your analysis in the discussion section.
Hi professor, i saw your earlier response on how to obtain the enzyme activity/ 1 g of crude, but how do you obtain mg of LDH/ g of crude?
You will have to make an assumption for this calculation about the elute fractions protein amounts and what exact protein they represent.
I have a few questions regarding LDH report. (1) When we're determining the total protein concentration for the elute fractions, do we also add up the enzyme along with the protein because LDH is also included as protein? (2)Do we need to show duplicates or just averages +/- delta in the purification table? (3)For the elute fractions, Im not sure how to go about reporting the error for the purif.table since only the 2 peak fractions were duplicated. Thank you so much.
(1) I don't understand your question. The protein assay detects the presence of all proteins. For the elute fractions, theoretically, the only protein in the elute fractions is LDH.
(2) Just averages and delta. (3) You can just use the delta for the entire total elute fractions since they make up the most error for these final total values. The fractions contain the most amount of enzyme.
regarding figure 1, for the sample calculations, can we present any calculation or does it have to be for the crude extract, that is, im presenting the calculation of protein enzyme concentration for one of the peak fractions, is that ok, or does it have to be for the crude extract? thank you
Chronologically, which section are you presenting the data first? It would be the crude, and so it would make sense to discuss it first. You could use the 40% sup as another option, but in the end, your organization of describing the steps in a chronological fashion would be really complicated and the reader would have a hard time following your steps. Confusion is what you want to really avoid in conveying your ideas and conclusions.
Dr.Kim, I'm doing my elution profile graph. My enzyme conc. looks like fig 23 but then since the enzyme concentration is much higher than protein concentration, my protein concentration did not show up. I'm concerned with my protein value since in load fraction, the conc. only ranges from 1 to 2. In fig 23, it seems there should be a lot of proteins in load fraction. Do you think my value is reasonable? Thank you.
You will need to set two axes so that you can visualize both values. See my previous responses on how to use excel for this.
Hi Dr.Kim. I was looking at table VI in page 43 of the lab manual and i was wondering what is the purpose of lane 1,2, and 3. Lane 4 has all the proteins shouldn't that be enough to compare Mr? Also i think you didn't mention anything about bromphenol blue in the lecture but just incase what is the purpose of putting that in our sample buffer? Thank you
Hi Ellen,
Lanes 1 through3 are for identification of the standards in case standards are hard to decipher or separate. As for the bromophenol blue, that is the loading dye that allows you to see that the gel is running in the correct direction and tells you when you can stop the gel. The timing of stopping the gel is important so that you separate out the proteins sufficiently in the resolving gel, and don't run the protein bands. The bromophenol blue is a reference for this. It is smaller than protein, and one can run empirical experiments to see how long a gel must be run relative to the dye.
Dr.Kim, for the elution profile in final LDH report, since we did not perform the enzyme assay for load and rinse fractions, should we include them in the elution profile? If we should, then do we just enter zero for the enzyme concentration? Thank you
Hi Sena,
For the enzyme, you will enter zero for the load and rinse (if they had zero activity). Now, you ran an enzyme assay on those fractions...it's just a qualitative one. I hope you know which assay it is. In addition, you should indicate on your elution profile that assays for those fractions gave zero activity by this qualitative assay. A little blurb in the figure legend the elution profile is fine.
When we're making the elution profile graph, do we plot the "total amount" values in raw data table for both protein and enzyme?
You are to plot the undiluted concentrations, not total. This helps to see any concentration effects that the column causes.
Good Evening Dr. Kim I am having deciding whether I should multiply my value for total amount collected in fraction for the 60% pellet by 2/ Should I do that? Also, should I also multiply by two when doing the same calculations for my fractions to take into account the other pellet? Tahnk You!!!
If you are assuming the 60% pellet (which you should), then you should multiply 2 to the column fractions as well. Simply put, you have another intact pellet that should yield the same amount for the 60% pellet and column fractions if you went ahead with assaying them.
Dr Kim, I think you said that we should include error bars in our elution profiles for our duplicates (am I mistaken?), but I can't find any way to include discrete error bars for single data points (it only adds one value of error bars for an entire series). Was I originally mistaken, or is there some way to add the error bars for single values? -Thanks
HI H,
You can list error in a table. For individual data points, there is a way in excel, but it is tedious and you have work around the rigid excel chart wizard. FYI: other graphing programs are typically used for science pubs such as Kalidograph, or Sigma plot.
Professor Kim, I have some questions on the final LDH report, my group didn't make duplicate for the protein assay for the 60% cut in the LDH lab 2, so we planned to assay the duplicate in lab 3. Then in LDH lab 3, we realized we lost the 60% cut, so we made another 60% cut from pellet 2, and protein assay the duplicates for that 60% cut. Now I have 2 data for duplicate in enzyme assay from pellet 1, 1 data of protein assay from pellet 1, 2 data for duplicates in protein assay from pellet 2. I don't know how to report them. Can you give me some advice? Thank you very much
I answered a question like this in a previous response. It's a bit ways back...but you should get your answer from this.
For the report, should we include a raw data table that we've been working on (which includes all duplicates with +/-) as table II and table III or would it suffice to make the tables in the format of table II on p125 of the reader and report just the averages with delta? Thank you.
It is easier for the TA to evaluate the raw data individually than seeing only averages. And it makes more sense since you will inputting only the raw data from the printout than a calculated average in which mistakes could occur in some instances.
Hi Dr. Kim, I'm making a bar graph to demonstrate the purity of LDH. However, my biggest SA is ~620 and smallest is ~1, so when I plot the bar graph, the smallest bar doesn't show up because it's too short (okay...I know my language here is not very technical, but hopefully you get what I'm saying). Is there anyway to manipulate the y-axis so that all of my bars would be clearly shown on the graph? If not, is it okay to present such a graph with "missing" bars in the report? Or should I just use a table? Thank you!
Hi Aradia,
The graph is okay, but only if you full duplicates for each of the elute fractions...shows overlap. A table is fine in this case, because you do not have duplicate values, especially for the smallest SA fraction.
Hey Dr. Kim, In calculating the final specific activity of our eluted fractions, do we just do it for our top three that contain enzyme in it? And then do we just add up the ammount of protein and enzyme in each fraction in order to get our final specific activity? How would you advise for use to calculate the delta? Thanks
See my previous responses...I think its about two responses back. ;)
Dr.Kim, for figure 1 on LDH lab report, how do you want us to put the sample calculation? Should we write the sample equation on the figure? Or should we write it on another sheet of paper? Thank you.
You can write it on the printout or provide another sheet. Whatever is neatest for this figure.
Hi Prof.Kim, I'm not sure about constructing the affinity column fractionation table (table III). Should we include all the fractions (load,rinse,and elute) or just the elute ones? For the highest peak fractions, we did those fractions in duplicate. Should we find the mean and delta for those in duplicate?Second question, for table I (final purification table), it says in course reader that we need to add up the entire amount of elute fractions. My question is for those highest peaks that we did in duplicate, would you suggest us to multiply the mean by 2 or just add the two duplicates? Thank you so much.
Table three should have all fractions, and for the highest peak fractions, deltas and mean should be given. Now, there is no reason to add the duplicate peak fractions twice. You need to only use the mean to add to the other elute fractions containing enzyme. As for the final delta, you can use the deltas from the duplicate fractions as your final delta since that delta is the largest contributor to error (the highest units of activity are in the top peak fractions making it the most significant fractions in the final value and error).
Hi. I was reading pg 43 of the lab manual and i noticed on table VI lane 6 has "Column LDH (2x)" Which fraction is this? Are we going to use top 2 peak fractions or just 1? Thank you
The 2x is twice the amount of LDH than what you normally use. This is necessary in case the normal 1X amount is not enough to visibly see your LDH.
Hi Pro. For the results section, you stated that we should explain the rationale behind each step. Do you also want us to explain how each step works? For example, how affinity column works.
Keep it as brief as possible. The affinity column does have many steps, but if you discuss that you will use AMP to separate AMP binding proteins from non-amp binding proteins, and then adduct for its purpose, that should be clear enough to the reader (given that the reader understands basic principles of column chromatography and knows what LDH binds to given by your background in the introduction).
Hi Dr.Kim I'm a little confused about in how to calculate the units and mg of purified LDH recovered from a gram of crude extract.... Do we take the purified amount divided by the crude total protein amount? And for enzyme take the purified activity divide it by the crude activity? Thank you!
Hi Ellen,
When you divide the crude mg from the units of pure enzyme, that will automatically give you a value of the units of pure enzyme per 1 mg of crude protein. You then just need to convert the value to units per 1 g.
Dr.Kim, for table II on course reader pg 125, it separates the data for 60% sup and 60% pellet. I think for LDH lab 2, we only needed to give the data for 60% cut. So, does 60% cut belong to 60% sup or 60% pellet? I'm confused. Thank you.
You should have the 60% sup data in there so that if you need to comment on the values in the dicussion, the TA can look them up and see if your assessment is valid.
Hi Dr Kim, as a follow up to my previous question regarding plotting the double y-axis on excel, do I highlight and graph the x-axis values and only the first set of the y-values FIRST or do i highlight all 3 columns from the start? Also, what is the enzyme data series that you were referring to? Sorry if Im being a little dense...
Highlight all three columns. Then when the graph is prodcued, double click the enzyme data series. And don't worry about the questions.
Hi prof, are you going to take off points for going over the limit of each section for the final LDH report?
Yes...we will. The only time limits can be passed if there major deviations to the normal procedure, and further explanations about troubleshooting issues had to be considered both in the materials and methods, results and discussion.
Hi Dr.Kim, I have questions about the protein assay value of my crude and 40% sup.For both crude and 40% sup, we did the protein assay once in LDH lab 1 and twice in LDH lab 3. Although we get lower value of the total protein and total activity of 40% sup compared to crude after we calculated mean and std.deviation(as expected), the individual data is weird.For example, when we assayed the crude and 40% sup in LDH lab 3, it turned out that in one data,the microgram of protein in 40% sup is higher than that in crude. I am not sure on how to explain this. Would you please give any suggestions? Thank you.
Hi...*stuffed from left overs*
It may simply be a precision issue. Do your error ranges for the specific activity and purifiction factor overlap cause the numbers of crude and 40% to overlap. If so, then this is an issue in which the assay needs to be done more reproducibly or you need a better assay to detect these small changes in the protein amounts.
HI, Prof. Kim. Regarding the decrease in the SA again. Our 60% pellet dissolved completely in the buffer, so there was no enzyme activity in the 60% sup (67fold dilution was used). Then probably enough salt wasn't added to precipitate the enzyme in 60% ammonium sulfate step?
Hi Lisa,
The dissolution of the 60% pellet really has no relationship to the 60% sup activity. And as for the 60% activity, you did not test any other lower dilution factor to see exactly how much enzyme you had. I would think it is hard to say the not enough 60% salt was added. That would imply that you should have add activity in the 60% sup. So, you are left with finding a model that would suggest a greater decrease in enzyme activity. Now loss of activity is not solely from losing LDH to various fractionations in the procedure. It could also be due to a loss of enzyme structure, which in turn affects function. Can you think of anything that could cause the enzyme to loss function, but no affect protein presence from coomassie binding.
Hi professor, for the filtration step, we didnt use glass wool, but i think filter paper (?) instead. is this a deviation that should be mentioned in the materials and methods section? thanks!
Yes...that is a deviation. It should be mentioned.
In the lab manual, it says for the % yield, adjust by a factor of 2 to account for the fact that only 1 60% pellet was used, but what if we had to add a small amount of the dissolved 60% pellet for some of the assays? Would that change anything?
That small amount of 60% pellet that was used for assays is included in your yield. It is lost for the column fractions, but you need worry about that loss. Basically, if you were to resuspend both pellets, and run the experiment in double the capacity (take 400 µl from the 60% pellet) you would still obtain twice the amount, lose the same percentage between the 60% pellet and column step, and obtain twice the amount from the affinity column. So it does not change anything.
Hi Prof.Kim, I was reading lab manual pg 38, about the sds page and I was not sure on the SDS-page. It says that determination of subunit is by addition of SDS. I understand that addition of SDS affects shape and charge of proteins (make them more similar to each other??)..but then, how does affecting shape and charge of protein enable us to determine the subunit ? Could you explain this please? Thank you so much.
Addition of SDS to protein samples allows us to determine the subunit Molecular Weight. I don't believe it states number of subunits. You need the native molecular weight. As for how it does this...it is not SDS alone, but you also need beta mercaptoethanol as well. This is to ensure the the subunits are broken up individually from each other.
In the instructions for preparing the LDH, we are supposed to explain whether the profile shows that the column was successful in increasing purity, and whether pure enzyme was produced. It is also suggested that additional tables/or graphs can be used to demonstrate the purity of the enzyme. Can you explain how to approach this. Shouldn't the elution profile and the overall purification table be sufficient to explain the purity of the enzyme?
Hi Lauren,
Well, does the profile give you exact values of specific activity for each individual peak fraction that can be read by the reader (assuming the reader does not have the appendix handy). Does the purificaiton table tell you individual specific activity values of each of the elute fractions that contain enzyme? Do some of the elute fractions have different specific activity values?
how do you account for the decrease in specific activity? our group has a lower SA in 60% cut that means we lost enzyme and obtained more protein? how does such a thing happen?
Hi LIsa,
Based on numbers alone, this situation would suggest that more enzyme was lost than protein. You will of course need to be sure that there is no reason to doubt the reliability of the data. And if the data is reliable, then you have to consider what could theoretically cause this problem. Did you add enough salt for the 60% step? Does the 60% sup contain enzyme? It is difficult for me to comment without seeing your data and discussing more...but I hope the questions I asked you give you start in thinking about what could have happened.
Hello, Concerning the LDH report, where does the rationale behind the experiments belong? In the introduction, results, or discussion? Thank you.
The brief rationale explanation is in the results section where it should be efficiently incorporated as you describing each step and result. Take the 40% step: you could describe the rationale as easily as just stating the purpose and effect. In order to remove the low soluble contaminants, we added ammonium sulfate to a 40% saturation and obtained "% yield" and "Pf". The nature of your understanding of the rationale will become apparent in your discussion section as your analyzing the various steps, sensitivity or limitations to the purifications steps, etc.
Hi Dr. Kim, Since I'm in the section that isn't doing the Gel exclusion until week 10 and so the protocol for it isn't due until then, what protocol do I have to prepare for this Tuesday's lab (week 9)? What lab is my section doing this Tuesday? Thanks. Happy Thanksgiving!
Hi Yasaman,
if you are in lab 1077, then protocol is not due till week 10. Your protocol will be then of only the SDS-PAGE lab.
can you explain what is required for figure I, do we use one sample protein and enzyme assay and label them? Can you also further elaborate on how to do the calculation that is required for part A in the discussion.
You will use the printouts of a protein assay and enzyme assay that includes the crude data. As for the calculation, you need to determine how much pure enzyme units is recovered for a gram of crude protein. So purification table should provide all that information. You did not start with a gram of crude protein, but if you did, how much pure enzyme would you obtain. You will need to make a new ratio of units of pure enzyme to crude protein.
When citing the lab manual for our LDH report, should we put Harold Martinson as the author? That's the only name in the beginning.
Yes.
Should we include spectrophotometer instructions in our materials/methods for our LDH or could we just say used spectrophotomer?
Although we did use new spec. instructions that is not in the manual, you may just refer to the manual which will cover all steps and spec. stuff.
Hi Dr. Kim. Do we need to show all the calculations for purifcation table and assays or just a sample calculation? And also, for the fractions where we did not do duplicates, how many sig figs should our final answer contain for the table?
Hi Sarkis,
Sample calculations are the only ones you need. The appendix tables will show your steps in calculations in which we can check for an math mistakes. For the fractions with no duplicates, these numbers are added to the other fractions with delta and will not make up the majority of the final value since they are not the peak fractions. You can adopt the delta for the peak fractions for the final total of elute fractions. The error will not be significantly different from the final value.
ive looked everywhere and cant find a "selected data series" option on the format menu. please help!
You need to plot your data first on chart wizard. After you have finished with the plot, double click on the enzyme data series to obtain the format menu. In that menu, you will find axis options, and secondary axis. If you are still stuck, e-mail for specific quesitons or see me Monday from 2 to 3 pm.
dr kim, for our ldh paper, when we present the tables can we do as many tables as we see fit, and number them as needed, as long as we convey the right information? or do we have to follow the numbering suggested in the instructions (pg. 123)? also when we include the prinouts obtained during lab, do we need to number them and refer to them during the report? hope my questions make sense. thank you!
You should refer to figures and tables in your text portion when necessary. This especially important in the results section. As for adding tables or figures, it is allowed when you really need to have an additional table or figure. It usually applies to those who may a unique data problem. If you feel you need an additional figure or table, you should tell me about the type of table or figure before incorporating it in your paper...or ask your TA.
Dr.Kim, I have some difficulties in reporting my data for the final LDH paper. During LDH lab 2, we assayed the protein assay of 60% cut and we planned to make its duplicate during LDH lab 3. However, we lost the 60% cut. Our TA told us to use the other pellet to make another 60% cut. Thus, during LDH lab 3, we used the second pellet to make 60% cut and assayed them in duplicate. My question is, now we have three values which of course the first one had the most protein (due to degradation, I think). Which two should we use? Should we use all values? But then the two of them come from different pellet. Second question, should we still multiply the value by 2 considering we used both pellets?Help! Thank you so much for explaining this.
Your situation allows you more information about 60% pellet. You can actually determine the true total if you find the singlet measurements of the first 60% pellet to be reliable. In addition, you will have the exact distribution of enzyme & protein from one pellet to another. So, when you are obtaining the total column fraction data, you should mutliply from a value that represents your distriubtion in both pellets...not by two. For example, if you have 45% in one, and 55% in the other, you ran the column fractions from teh 45% pellet, then the column fraction data should be multiplied by 100/45.
Prof.Kim, for the protein assay of crude extract and 40% sup, my group did only one measurement in the LDH lab 1, but then in the LDH lab 3, we made again the protein assays of crude and 40% sup in duplicate. So now, we have three values of each sample. One from LDH lab 1 and duplicates from LDH lab 3.Can you give suggestion on which values we should choose? Thank you.
Hi Dee,
If there is no reason to remove the other values, then you should include all three.
At what time and where will your office hours be this monday? thank you!
From 2-3 pm, in room 1089.
hi professor, i'm a little confuzed i thought in my lab section last week it said that the final LDH paper was due the day of lab but on the schedule online it says Monday the 28th by 5:30. which is it?
It will be due in seven days. So for Tuesdays lab at 5:30, it will be due Monday at 5:30 in 1060. We do not want it due during your lab as you will find that most students will not be very "awake" during the lab and may be more prone to error.
Regarding the Final LDH paper, do we include the printouts of our assays or should we summarize the data in a table? What should we included in the results section as far as tables, graphs, charts, etc.?
Hi Frans,
You will need to include all data printouts in the appendix, and you need the talbes. As for the result section, see instructions in the course reader.
Hi. Do I need to compare top 2 peaks specific activity to determine the purity of the 2 fractions for results section? Or do i need to compare all of the fractions that have LDH? I know that specific activity should be the same for both in order to conclude my fraction was pure but how close do the values have to be? Thank you!
Hi Ellen,
You need to compare all that have enzyme. As for how close, consider the error in your measurements as one way; error of duplicates or error in the spec. If they are significantly different for some, then you will have to suggest why that's the case.
Should we factor in volume changes in the column fractions due to aliquot removal for assays and if so how?
Hi Josh,
Factor volume change for what reason? For total protein and enzyme amounts - you take the total volume for the fraction (before aliquot removal) which is similar to all other purification steps. If you are referring to something else, please let me know.
For LDH Lab 3, are we running enzyme assays in duplicate on ONLY the elute fractions with +++ or will we also run an enzyme assay on their faintly-colored/uncolored neighbors that appeared on the spot plate next to them? Do we have to repeat protein and enzyme assays on these faintly-colored/uncolored neighbors if the original data was suboptimal or are they not that imnportant?
You will run enzyme assays on all elute fractions that have a plus and the adjacent fractions with no activity or little activity. Only the two top peak fractions will be run in duplicate. As mentioned in lecture, you need to repeat P.A. and E.A. on data that is suboptimal for the assay. To clarify, run a better dilution for the assay, but if you end up estimating that the 1:1 dilution is the only one left to run, and it turns out to be suboptimal, then you can stop with that fraction. However, non-enzyme containing fractions are a lower priority and so if time runs out, at least you have the all important elute fractions completed which is more important for the conclusions, paper, and future experiments.
hi dr kim, for our LDH 3 protocol, would it be okay to have everything we need to do summarized in an excel chart, showing what DF needs to done and what volumes to use, etc, instead of explaining or writing out the procedure steps? Thank you.
Yes...that would be great and perfect for having your group members be completely organized at the task at hand.
Hi professor, so for this weeks lab all where doing is Assay of the affinity column fractions and just catching up with everything?
Yup! Seemingly easy, but could possibly take forever if you and your group do not carefully prepare and perform the assays.
Dr.Kim, A little trivial question: Is spot plate assay a qualitative or quantitative analysis? It measures the intensity or the concentration of the enzyme in a relative sense, but it cant tell the exact amount.... (?!?!) Thanks.
Hi AEO,
The spot plate is qualitative measure relative to the enzyme assay. You are subject to much greater error in measuring the intensity by eye, as well as the assay itself is not well timed from sample to sample.
Professor, for LDH Lab 2 Second Progress Report, do we have to calculate the total protien amount for the fractions we did the protein assay on? Some of the fractions for some diltuions had absorbances below 0.3 while some were within the target range...should we still fill in as much as we can for the fractions data on the Purification table? Or do we wait to do this in the next lab report after LDH Lab 3?
You should still include all values that you have. It will help get an idea of the current status of your data so you know if there is any additional experiments that needed to be run in addition to redoing the suboptimal protein assay values, and your strategy in calculation can be evaluated by the TA to look for any errors.
For the LDH final report, we are given certain prescriptions for how long each section should be(i.e. 1/2 page), do these prescriptions apply to before or after we double-space?
The page lengths apply to double spaced texts. I know it will be rather difficult but part of scientific writing is to write comprehensively and concisely which as some of you may noticed during the exams is very difficult. Unfortunately, you will be working against entropy. So as you are putting the paper together, you should write out your ideas and then seek to condense your writing. The only area in which we will have some flexibility in the length is the discussion, especially for those who have encountered troubleshooting issues that need to be explained. Of course, that is not a license to write an epic novel at the end of your paper.
When we calculate spec.activity, should we divide the table (rounded) values of tot act over tot prot, or should we divide the values before the rounding? if i do it before rounding, the spec. activity decreases going form 40% sup to 60% pellet. if i do it after rounding, it actually increase. Also, what reasons could there be for the specific activity go down going from 40% sup to 60% pellet? thanks for your help/
You should never use rounded values for intermediate calculations. Simply put, the numbers you determined from dividing rounded values are not accurate and cannot be trusted. But when presenting total protein and enzyme in the purification table, the presentation of those numbers need to be in sig. figs. and rounded appropriately.
hi dr kim, in the progress report form, it says to run duplicates only for the 3 fractions which had the most LDH. Does that apply to only the enzyme assay or to the protein assay as well? thank you.
The TAs will make another amendment to that procedure. You will run duplicates for only the top two peak fractions (both protein and enzyme) unless you cannot distinguish the peaks between fractions in which case you will have to run duplicates on three fractions.
hi professor. for the lab2 progress report, do we need to include the raw data table? can't we just show calculations separately below the purficication table?
Hi Jae,
The raw data table helps when you are using excel. And you should use excel to minimize any mistakes that you might make. You are more prone to make calculation errors when you are inputing values over and over again, as opposed to having formulas in an excel file that can be copied and pasted for various purification step samples. In addition, numbers can be carried with greater ease from calculation step to another.
i was just wanted to confirm something with you regarding the raw data table. do i multiply all the 60% pellet data by 2, including the enzyme assay, protein assay and the column fractions?
Yes...but multiply the total protein and total enzyme only. Do not multiply concentrations because you are not necessarily increasing concentrations if you assayed both pellets.
Professor Kim, Can you give some directions on how to estimate the dilution factors for the enzyme assays of the elute fractions from the 60% pellet enzyme assays and the initial protein concentration assays of all of the elutes? Thanks
Your 60% pellet enzyme assays is the reference for the dilution factors for the elute fractions. The 60% step represents the initial amount of enzyme that will be spread out in the various elute fractions. So assume that 60% is 100% of enzyme. The elute fractions will have percentage amounts of it. Who will you know what percentage you will have? The initial protein assays should give you some idea of how much enzyme is in each. If the column is done correctly, the protein in the elute fractions should be LDH. So the total protein amounts will have proportionately equal enzyme units of activity. Use the percentages to determine how the dilution factor should be adjusted. But you also must adjust the dilution factors by volume change. There is concentration affect by the column (5mls of load into a 1.5 ml bead layer). Thus, the volume decreases for each fraction and that must be accounted for as well. It is difficult for me to explain this over the web so that you can figure it out on your own. You may want to meet with me later this afternoon (after 3pm) or see a TA.
Prof Kim, for LDH lab 2 report, should we make our elution profile to analyze the column fractions? Thank you
You would only have your protein data, but the more you include, the more feeback you can get from your TA as to whether you care creating the profile correctly. It would only help you for your final report.
Hi. I am not clear about significant figures in the purification table. You mentioned "the range in which conclusions can be made" but what is this range?
The range is related to how you will make conclusions regarding yield or purification factor changes. For example: compare you crude to 40% values. Do you find that the purification factor change is significant or certain, or do your error bars overlap? You ability to make a conclusion about any increase or decrease of purity not only relys on the comparison of the factor values, but also the error bars and if it causes the values to overlap which would make any differences between the two values inconclusive.
Professor Kim, for LDH lab 2 report, the instructions says that we should include calculations. Could you explain which part of the table that we should include calculation? Because in ldh lab 1 report, i dont think we need to include calculations. Thank you.
You can continue presenting your raw data table as your calculations. But you should indicate when certain values are doubled based on the splitting of the pellets.
Hi Professor Kim, I have questions about LDH lab 2 progress report. It's said in the instruction that we should multiply our % 60% cut yield by 2. It makes sense to me. But then, I'm wondering when calculating purification factor, should we multiply the specific activity of 60% cut by 2? Could you explain this please? Thank you.
Hi Dian,
You only need to multiply the total mg or yield by two. That would be sufficient in assuming twice the yield. Would it make sense to multiply the specific activity?
Hi Dr Kim. In our purification table, do we report 40% salt and 60% salt separately in our purification table?
Yes.
Professor Kim, I was unclear as to when the actual LDH paper was to be turned in. In our lab reader it says that it is to be turned in at the beginning of next lab, but we have yet to assay the enzyme from our column fractions, which we need to complete the purification table. Are we turning it in at the next lab or is it actually do another day? Thanks
Refer to the due dates of reports on the VOH home page. It will be due at the start of the gel labs, not during LDH LAB 3.
in a previous question regarding sig figs in the first progress report, you said that we should not be docked points for sig figs. does it also apply to the purification table, in that they are also incomplete data?
The purification table (which is not the raw data table) must be reported in significant figures which will provide you and the reader the range in which conclusions can be made. As for you stating incomplete data, what exactly do you mean?
Just wanted to know what we needed for the LDH lab 3 protocol. Is it just repeats of any data that is not adequate and the enzyme assays of our fractions?
Hi Juniou,
Yes. Be sure it is well organized and detailed with all the pertinent information: exact dilution factors, volumes in how to make the dilutions, number of substrate tubes, and protein assay samples.
Hey Professor Kim, I was just wondering if you would be around during tomorrow's lab or still have office hours. Thanks
Yup. I will be there. I will primarily in the labs and if I'm not there, I will be in my office (or the bathroom).
Professor Kim, Should p.34 part E in the lab manual be included in LDH 2 protocol? Thanks
Portions of the protein assays for column fractions should be in the LDH Lab 2's protocol.
In the first progress report, I was docked points for sig figs on my raw data table. This aspect confuses me about the raw data table, we are 'presenting our data in a report' but it is also supposed to be 'raw data'--does this mean we should adhere to sig figs presenting rules for final reporting, or show as many digits as possible so the TA can follow our calculations/raw data?
You should not be docked points. It was an error in grading. Please see me to discuss this further.
If one of my dilution calculations came out to an odd number like 69-fold, can i round it to 75 or should i do the 70-fold?
You can round to 70 fold. Keep in mind that reliable data is a range in both assays. Hopefully, you will hit the range in your first try.
hi dr kim, for the protein assay on the 60% cut dilutions, do we use the same method for finding the D.F.? For example, if my DF for the 40% sup gave 20-fold as optimum range and its volume is 50ml, 20 x 1/3 x (50ml/5ml). Is this the correct way to do this?
Looks good. I would decide on a range of dilutions that include what you calculate and something lower - just to safely get in the range of protein assay standards.
im a bit confused about ldh lab 2 procedure. do we just run the protein assays on the load, rinse and elute frations or do we have to dilute it accordingly this lab?
You should always make dilutions of your unknown especially if you know that it will be too concentrated for undiluted fractions. Now, the question is whether you know if it is too concentrated? Your 40% supernatant most likely gave an adequate absorbance at 25 fold. Consider what will happen to the 60% pellet. The volume decreases and the amount of enzyme is a third of the total. So the 60% pellet's protein can also be looked at in a similar fashion as enzyme concentration changes, but with an understanding that we are not entirely certain how much protein is collected in the pellet. So if you used a 25X for the 40%, then the 60% pellet will require 25xVol. Fold Change x 1/3. The volume fold change is about 8-10 fold depending on your volumes. You will see that you definitely dilute the 60% pellet to get adequate protein concentrations. So what then will happen to the concentrations in the column fractions, and what dilution factor will be appropriate. Try this out to determine which dilution factors you will test. If you are stuck, look over lecture 6's notes to go over the logic for dilutions, and you may need to diagram the column procedure to see what kind of concentration changes may occur.
in the same question (fall 04 Ex 4a), why are there only load and elute fractions and no rinse fractions? are we to assume that the rinse fractions are part of the other fractions? thank you.
Hi Gina,
there was no mention that were no rinse fractions. When looking at the given profiles, the pattern of elution of load, rinse and elute should be understood. The first few fractions would be the load in which the contaminant proteins that do not bind to the column, elute. The mid fractions would be the rinse, where the contaminant proteins are completely washed out of the column. And the last fractions are the elute. I'm not sure how you thought there were no rinse fractions. Maybe you could explain this to me so I can see where the confusion could arise in the question.
prof.Kim, for the past exam 4 summer 05 question 1b, I think the question is very similar to the spring 05 question that we went over in the review session tonight. I'm wondering why I cannot use the method that is written in the spring 05 one. So, what i did was i divided total unit of (20-50% cut) which is 1500 units to total unit of 20% sup (which is 1550 units). The result is 96.7 % NOT 90.9%. Why is that? Am I missing something? Thank you.
Hi Dee,
There is a math error in the spring 05 which I still did not pick up during the review. It is 96.7%. You're calculations are correct.
Hi prof kim on #2a of fall 2004 exam 4, i dont see why non-amp binding proteins would move faster than LDH when adduct is added without the rinse step. Because there is adduct in the buffer, wouldnt LDH travel faster along with the buffer than the non-amp binding proteins when the adduct is added?
Non-amp binding proteins always remain in the mobile phase at all times because there is no interaction with AMP, whereas LDH can still bind to the stationary phase. Adduct does not remain constantly bound to LDH - there is always an equilibrium of binding and release when you consider non-covalent interactions.
Hello, what is the purpose of the rinsing with buffer step in the column chromatography? Is it to wash out any residual non-AMP binding proteins that may still be in the column? Thanks.
Yes. Given that there is still a 1 ml of load still in the column at the time the rinse buffer is added, the rinse is required to allow for those non-AMP binding proteins to elute, and additional buffer is also required to remove any weakly bound proteins as well.
exactly up to what section in E.B. will we be doing for LDH 2?
Read up to the end of page 34 to find out. Keep in mind that if you understand the column procedure, you will know which assays you can perform in LDH LAB 2 versus which assays you will have to perform in LDH lab 3.
Prof.Kim, I have questions about the different pH of buffer used for this experiment. So, we're using pH 6 buffer for resuspending 60 % pellet AND we also rinse the column with pH 6 buffer as well right? If it's right, then why on exam 4 summer 05, question 2b, it says that "the adduct at correct pH". Also, we only use ph 7.2 when diluting aliquot right?
All solutions involving the column are at a pH of 6.0, and that includes the solution containing the NAD-pyruvate adduct. And you only use pH 7.2 buffer for the dilutions used for enzyme assays and protein assays.
Why is it bad to use pH 6 buffer for 60% pellet and elution fraction bad? Is it because it might increase the activity of LDH and there would be an overestimation of our enzyme concentration?
pH 6 buffer means more protons in solution (relative to pH 7.2 buffer). This increases the protonated state of LDH, providing a more stable binding to substrates. A more stable binidng to substrate means less release of substrate/product from the enzyme. This means lower activity by the enzyme which would be a DECREASE in activity if we used this buffer for the enzyme assay.
Why is it bad to use pH 6 buffer for 60% pellet and elution fraction bad? Is it because it might increase the activity of LDH and there would be an overestimation of our enzyme concentration?
pH 6 buffer means more protons in solution (relative to pH 7.2 buffer). This increases the protonated state of LDH, providing a more stable binding to substrates. A more stable binidng to substrate means less release of substrate/product from the enzyme. This means lower activity by the enzyme which would be a DECREASE in activity if we used this buffer for the enzyme assay.
Hi Dr.Kim, I know that you said for section VI E pg 34 lab manual, we should start for the protein assay on this week lab period. But then, are we responsible for being able to do the protocol item of this section in order to do well on midterm number 4? If yes, please help. I'm not clear on finding the dilution for each fraction.I know there is some guidance on the reading but I'm still not clear. Can you give some hints pleaseee? Thank you.
You can actually determine the approximate range of dilution factors for the protein assays of the column fractions which should be part of your protocol for this week. You must first remember that most of the protein loaded on to the column are contaminant proteins. So, using the elution profile in the lab manual will make sense. The load fraction should have a similar protein concentration to the 60% pellet. And all other protein concentrations should be relatively lower. But note that when you test for protein amounts, you will still need to test a range of dilution factors. So you will most likely two different dilution factors for each fraction. All in all, the guidance should make more sense to you in how those dilution factors are chosen by the lab manual. For you to come up with those dilution factors, you would require some initial assays on the 60% pellet which you do not have yet.
Hi Dr.Kim, I have questions about the ammonium sulfate questions on pg 52 course reader. For #2b, I was able to get the right answer for finding the SA of 40% pellet. But then, I couldnt get the right answer for the SA of 40% sup.I notice, for the sup, we just diide 600/3100 and NOT include the previous steps. What is the reason? Does this apply when finding the SA of sup for single step of for two steps procedure as well? Thank you
The listed values for the supernatant is the amount of protein and enzyme left in supernatant at each survey step. As you precipitate protein and enzyme, you are left with less and less protein and enzyme. There is no reason to add previous supernatant steps.
Hi Professor Kim, For our chromatography, provided that our LDH is not saturating and no LDH comes out during the rinse phase, how do we know when we should stop the rinse phase and move on to the elute phase? Since the spot plate only assays for enzyme activity and not contaminating proteins, how can we tell when the time is right for us to move on the elute so that we get the greatest purity and yield? (i.e. after non-AMP binding proteins have flown up but before any LDH has left the column.) Thanks!
You only know to stop by our suggested amount of rinse buffer we have prescribed in the procedure. The manual provides an exact volume which is 7 mls (0.5 ml twice and then 3 2ml rinse fractions collected). The only logic I can provide for choosing this volume is that a few mls is used to ensure that all non-binding proteins are eluting from the 1 ml bead layer, and a few more mls are used to be sure of that no other non-AMP binding proteins are present. If the bead is readily available, optimization of the rinse amount can be done to be certain of the removal of these contaminants, and that LDH does not flow out prematurely before the adduct. You would not generally know this without the empircal study.
What happens to the yield and purity for the elute fractions......if youre agarose beads dry out before adding the load? In one exam it said that both the yeild and purity would drop. But in another exam it said that there is no yeild and purity. Which one is the correct answer?
Hi Linda,
At least for this example, we know that if the agarose beads dry out, then much of the enzyme if not all will be lost to the load fractions. As for the answer, it is a frame of reference. Since we theoretically only use the elute fractions as the purified step, then there will be no yield or purity that could be calculated since there is not enzyme available in the elute fractions.
Can you please post histograms for exam 2 and 3? Thank you!
Hi Ellen,
Exam 2's histogram will be up soon. The were some wierd things with the file as seen in exam 1's histogram that will be corrected. Exam 3's histogram will be up either Friday or next Monday.
Hi Dr.Kim. On bottom of page 54 of the course reader it states that i have to consider the volume changes in the fractions. I'm not sure how this affects the dilutions. Since most of contaminates are in the load, wouldn't I make the same dilutions as the 60% cut?
HI Ellen,
For most of the load fractions, the dilution factor would be the same. For the first load fraction, it will not. Why? What is in the first load fraction? Hint: There is always buffer in the column.
In the LDH lab 1, I got higher percent yield for 40% Sup. What could be the reasons?
A higher 40% yield in the sup. Well, you have to consider all possibilities and evaluate to see which model is the easiest. One would be that it was pipeting error or the way you made your dilutions. Did you make replicate sets of serial dilutions? There could be random error. So you would need to repeat the runs in order to test this hypothesis. Which samples? If you have no reason to believe that either the crude or 40% sup is subject to more error than the other, you would have to assay both. Once you have exhausted this possibility and found that the yield still increases, then you have to consider another model. Something you may have to do for the final LDH report. So for now, when you go into the next lab, test for the random error first. Then, if that doesn't work, we can discuss the other models in more detail. Essentially, this is the process in developing models. Starting from the simplest model, testing it, and from that result, deciding on whether a more complex model is needed or not. We could already hypothesize other types of models, but these models would require assumptions that are pretty improbable. Such models for yours would be like enzyme is added to the 40% sup, which does not seem likely. And the other model would be enzyme in the crude degraded which you have to suggest that something happened to the crude that would cause this degradation in the sample crude aliquot. If you can think of specific events that occurred during your experiment (such as one group had a piece of ice that fell in), then you could hypothesize a model, but if not, first test the simplest model in the next lab.
Professor, for this week's lab protocol (LDH Lab 2), do we need to include section E (pg. 34) or will it only be section D (pg. 29-34)? THanks!
Hi Jenny,
You will include column fraction assays, but just for protein. Enzyme assays will be in the next lab.
Prof.Kim, when finding highest purity of ammonium step procedure, we always try to find the step in which the SA is the highest, right?And for finding the highest yield, is it by finding which step gives 100% yield? Thank you.
Yes...but for the highest yield, you still choose steps that provide an increase in purity (if the survey study shows such an increase).
hi prof kim, ive been trying and trying to figure out on the spring exam 4 #1b, how it is 90% recovered from 20%, and not 84.8% as was the percentage yield from part a. am i missing something crucial here?thank you
Hi Help,
It is an error. It should be 84.8% just as the answer shows in part a) of this question.
Prof Kim, for the calculation of survey experiment of ammonium sulfate addition, I notice that we never include the total enzyme of the first step. For example on practice exam spring 05 in the course reader, question 1A asks the total unit of enzyme for 20% to 40%, I notice that the total unit is 1400, not 1500, which means that the total pellet for 20% is not included. What is the reason behind this? Thank you.
When you perform a two step procedure, the first salting step discards the pellet. Thus, the enzyme is lost and not included in the final 2nd step pellet.
Prof.Kim, are we going to do the survey of finding which %step of ammonium sulfate works best in our next lab? Im asking this because there are questions of that in past exams. But then, when I look at the lab manual, it seems that there is no procedure that lead us to do that. So, are we just expected to be able to do the calculation of this survey? Please clarify. Thank you.
Just understand how the survey experiment works. If you understand how ammonium sulfate procedures work, then this problem should be easily doable. So its a good exercise to solidify aspects of this procedure.
Hi Prof.Kim, on page 33, lab manual, it says on step#3 that we should "obtain an additional tube, and calibrate it for 2ml, in a manner similar to step 1. I'm not clear on this sentence. Does it mean we're doing two column chromatographies? first with 1 ml and second with 2ml? Thank you.
The 2 ml guide tube allows you to know when you collect 2 ml fractions. You place the guide tube next to your tube that is collecting and you wait till the column fills the tube up to the same level as your guide tube.
Professor, in class you mentioned that more of the proteins that don't bind to AMP will be found in the rinse fractions because the load fraction will contain a lot of buffer that will is already in the column so this fraction's protein conc. is diluted by the buffer. My question is, #25 of exam problems part a states that load fractions will contain most of the protein. shouldn't it be rinse fractions? thanks.
I believe I have either misspoken, or you did not hear me correctly. Most proteins are non AMP binding proteins that elute from the column during the load phase and if anything, the first rinse fraction. Now the part about the diluted fraction is the first load fraction collected. It is diluted due to buffer initially present in the column. That ~1ml buffer is collected with the first 2ml load fraction. Thus, the first fraction is diluted by 2 fold.
Good Evening Dr. Kim I had a question regarding the first progress report. Do we have to do a table where we figure out the Purification factor, yield, and LDH activity or do we just construct the table that appears on the instructions for the progress report. Also, when determining the concentration for the diluted and undiluted solution the only difference is that for diluted we dont multiply by the dilution factor, and for the undiluted we have to multiply the units/ml by the dilution factor or the ug/ul by the dilution factor for the protein concentration,right?
Hi Cesar,
You need construct both. One table is for evaluating your raw data calculations that lead up to the final purification table. If you use excel now, and create formulas that you copy and paste for each fraction you analyze, this raw data table should be easy to construct and utilize (no rounding needed in this table). However, your purification table should show rounded values with errors. As for the concentrations, the undiluted and diluted concentrations do differ by the dilution factor. But becareful that you read what each value is asked for...such as units in a 3ml fraction...that is not a concentration. And the protein asked for is µg per aliquot. That is simply the µg of protein listed on the print out before it is divided by 50 µl.
Maybe its real late and I ve be studying too much bio chem but Im stuck on the first of the additional Ammonium sulfate questions: I've tried using ratios like this: ( x g ammonium sulf.) / (50ml) = 10% and get 5. But I know theres something I'm doing wrong here but can't figure it out. thanks for the help
It is late. But I will help you on this since it's something you have picked up before exam 3. The % saturation is not the mass percent to a volume. That is weight per volume. The percent saturation as defined in lecture 5 is the percent mass of salt relative to the amount of salt that gives 100% saturation. So, for an easy explanation: if 0.6 g of ammonium sulfate added to 1 ml of water gives 100% saturation, then 0.24 g of ammonium sulfate added to 1ml will yield a 40% ammonium sulfate staruated solution.
Hi prof.Kim. I remember in lecture and office hour, you were talking about the survey of finding the step of ammonium sulfate fractionation that give highest purity and yield. But I couldnt find any section in this week lab period in the lab manual that says that we're going to do that. I sense that it might be section VI E. But im not clear about the fraction number. Is it correspond to %ammonium sulfate? Please clarify this. I'm very confused. Thank you so much.
This section is in the course reader...I believe it is on page 54. It is not an experiment that you will be doing, but it is an experiment that you need to understand in order to know how the 40% and 60% saturation levels were determined for our use.
Hi amount in collected fraction, do we multiply the "unrounded" concentration in undiluted fraction to the total volume? or the concentration where the delta has been accounted for, so "rounded"?professor, for the LDH LAB 1 report, to find the total
Hi Jae,
For any final value that is to be reported, you should never round any calculation. The best way to do this, is set up your excel tables with formulas that give you the final total amoutns of enzyme or protein. You do not need to round values in the raw data table. Only in the purification table. But be sure not to use rounded numbers for calculating the specific activity, yield, or P.F.
Prof. Kim.. I was just wondering what part of the manual we should use for the prelab this week and what sections of the lab manual will be the topics for the exam on Tuesday. Thanks.
See the course reader - lecture 6.
Professor, are we doing part E on page 34 during this upcoming lab-period?
Only part of it. You must start the protein assays on the column fractions. It's the only way to get preliminary information for the enzyme assays without wasting tremendous amounts of time. We will discuss this Thursday.
Hi prof! What is the ideal dilution factor to assay activity of the 60% solution? If you don't want to tell me straight up, what are the criteria? I had everuthing written in my class manual but I lost it on Friday, and I don't want to be making mistakes... thak you prof! Pauli.
Hi Pauli,
Well, you know me so well now. The criteria is what is important and here it is: you have to consider volume change, and amount of enzyme change. The volume from the 40% to 60% step is a decrease. Which would be a factor for an increase in concentration. If you assumed that you recovered 100% of the enzyme in the one pellet you resuspended, and the volume decreased (due to precipitation), the concentration would increase by the fold difference in volume. So that means, the dilution factor that needs to be used is a fold greater than what was used for the 40% step. But you almost consider the decrease in amount of enzyme. You are losing 1/3 to the 60% sup. Which means that you are left with 2/3 of the total enzyme in the pellets. But it's half that amount since you split the pellets. So the dilution factor must decrease by 1/3. See if that starts you in the right direction. If you get stuck...ask me again. It might help to give me some specific values you have for your lab, and what changes you accounted for.
Hi. I was wondering if i should add crude extract #1 and #2 for LDH assay and record their average and delta.. that would make the table a little different right?
Hi Ellen,
Yes...you should add additional columns when you have duplicates. That is acceptable and should be done.
Prof., what was the mean for the lab practicum?
Hi jr,
I believe its 71 and 69 for protein and enzyme sections.
Hello Dr. Kim In the lab manual it says that we need to add .24g of ammonium sulfate for every ml of supernatant. During the review, I think I remember you saying that we were going to use about 11.2 g of the sulfate. How did you obtain this number? Do we have to guestimate on how much "stuff" we are losing from the homogenization to the centrifugation step when we obatin the crude extract? We begin with about 58ml, so does that mean that when we obatin ouyr crude extract we should have about 47ml left? Because 47ml*o.24g is about 11.2. Thank You.
Hi Cesar,
That number was for an example volume. During the review, I mentioned that IF we had 47 ml of crude, then when we performed the ammonium sulfate addition, we would need 47ml x 0.24g/ml = 11.2 g. Now, your volumes may differ, so don't expect to get 47 ml...may be more or less. As for the crude volume and its difference from original beef and buffer volume, you will need to recognize that volume is lost as you blend, transfer solution, centrifuge, and filter. You should consider the how the volume is lost and estimate what you think the final volume of the crude should be.
hi professor Kim, for the progress report for LDH lab1, we do not need to include the calculations in the report right? just the tables with the figures? thanks.
Yup! Just the tables, and your expected summary of analysis.
Professor for our raw data table (progress report 1), what are the rows labeled "Total volume" and "Total amount in collected fraction" referring to? Thanks!
Hi Jenny,
Total Volume is the volume you collected at a step before you removed the aliquot which is number you require to determine the total amount in protein or enzyme in that step.
Total amount in collected fraction is the total amount in that step. The protein and enzyme in the ammonium sulfate step or crude is a fraction of the original amounts in tissue sample.
Hi Dr Kim, For the 2nd addition of ammonium sulfate, shouldnt we add 12g/ml more, instead of 13g as stated in E.B.? Since we already added the 0.24g/ml for the 40% cut, which is 40% of 0.6g/ml, to reach 60%, I think it should be 0.12g/ml more, correct? Thank you.
Hi Gina,
It is 0.13 g for a couple reasons. One is that 100% saturation is not exactly at 0.6 g, and that masses are rounded to the hundreths place. Plus, there are few correction factors that does not make this calculation exactly to 0.12 g. So after rounding the true value, the 20% addition is actually 0.13g into 1ml.
i'm having issues with the nomaclature of this lab. i understand the difference in what is being measured in "total protein" and "total enzyme." but why do we use two words that can be used interchangebly in biology to indicate two different things?
You cannot use the terms interchangeably. Proteins are not necessarily enzymes. There are structural proteins and transport proteins that have no enzymatic activity. So an enzyme is a type of protein. And for this enzyme purification lab, the enzyme total we are referring to is LDH, and no other enzyme. So, the nomenclature is clear in the context of our project.
Dr. Kim, in your practice exam IV, question 3b is confusing. It asks for the protein concentration in the crude extract, yet the answer uses enzyme activity values to calculate it. I don't beleive that we should assume all protein in crude extract is LDH since it is a small percentage of total protein concentration. Please elucidate.
I want to answer your question but, which question are you refering (from which exam)? Also, are you sure its for exam 4?
for the practice question 6 in the lab manual, the solution says that enzyme assay needs to be done first to check if the recovered enzyme is in the appropriate proportion. What does it mean by "appropriate proportion"?. Does it mean the same as saying "to show that the recovered enzyme is in a reasonalbe range; not too low, not too high?"
Yes...it's the same thing. But to be clear...proportion refers to relative units in the ammonium sulfate step relative to the crude extract.
Hi Dr. Kim. I was wondering on the lab practical exam, how many points did the part that we had to show how we diluted the unknown have? thanks
Hi LD,
If there is a problem with your data and dilutions do not correspond or are not shown, then there will be a deduction of points. But for the most part, there is no deduction if your data and dilutions are consistent.
hi dr kim, what is the purpose exactly of the protein assay in LDH lab 1? is it so that we can find the protein concentration (part of purification table)? also, does the total protein concentration include the enzyme as well or just the bunch of other protein "junk" that we dont want? Thank you
The protein assay is to determine the protein concentration in each of the purification steps for the use of the purification table. The enzyme is part of the total protein amounts which contains both enzyme and contaminant proteins. As we purify the contaminant proteins away, the protein amounts should drop. By doing an enzyme assay, we know how much enzyme we have at each step. When we compare enzyme activity to protein amounts from one step to another, that ratio should increase, because there are less protein contaminants. Thus we have our purification factor from the comparisons of the two specific activity values.
From your previous Q and A you mentioned that one control must be assyed for new set of assays..That would be the pyruvate one right? Is it because crude and the 40% sup has different amount of protein? Also do we need to do the pyruvate control again for our duplicates?
Hi Ellen,
So the new control is no pyruvate control and you only need to test it for your crude extract. Why? Well keep in mind what it tests about the crude extract? Does the crude contain any component in there that will cause NADH to react independent of the LDH catalytic activity? We expect there should be no activity. So the crude would contain all the same components if not the full cellular components than the purified 40% sup. So there is no need to retest this control for the 40% sup since we gain no further information by this control.
Hi Dr. Kim, At the end of Friday's review session, you mentioned that you did not cover as much material as you wanted to and that you would cover the rest on Monday's review session. Will tonight's review session be the same as Friday's or will you cover more information? In other words, is it essential that I attend tonight's review session if I already went to Friday's? Thanks.
We covered pretty much about the same material. The latter material was just tying up the next steps - 60% step which I managed to preview enough. So you need not attend Monday's but be sure to look over the negative controls and the questions in the back of the manual and my previous exams.
Hey Professor, Regarding LDH Lab 1, do we collect the supernatant from both centrifuged tubes or just one?
Hi Ben,
you will pool your sups.
Prof.Kim, for the 2 negative controls of enzyme assay, I'm not clear about the second one( the one that says to substitute buffer for pyruvate).I'm not sure on how to do this. So, we dont put pyruvate for this control? But then, for the substrate tube, we add pyruvate and NADH, so i'm confused now. Would you please explain it? Thank you.
So for this control, you will replace pyruvate with buffer, but still add NADH and the enzyme aliquot from the crude. So, what will this control test...well we assume the conversion of NADH to NAD+ requires LDH and pyruvate. But could there be other components in the crude that could cause the change of NADH independent of our studied process. Keep in mind that controls typically have all the same components except for one. So the total volume and concentrations of all components must be the same for this control except for the precense of pyruvate (needs to be replaced with buffer).
Hi Prof.Kim, I'm sorry if this is not a useful question, but im really curious. In lab manual page 25, I was reading the second paragraph on that page about the protein assay that cannot detect all proteins equally. It says that it's undesirable. I am wondering why it's undesirable? I thought we need it to detect the proteins differently so that we can compare our crude extract to standard protein? Thanks Prof.Kim.
The protein assay is supposed to detect proteins to an equal extent. It is our measure of total population of proteins which will aid in our measure for purity (specific activity). It is our general assumption that coomassie binds to BSA in a similar mass ratio to any other globular protein - so that we can compare standards to sample directly. And for most proteins, that is true. But in reality, some proteins can have varying compositions of hydrophobic regions causing some error in the comparison of our unknown to the BSA standard (one type of protein). However this error, because we make relative comparisons from one step to another, is for the most part normalized out by this comparison.
Hi Dr. Kim, I have a couple questions for you. On TUesday's exam, will it only cover up to page 48 of the CR? The exam practice problems in the lab manual have a couple questions about column chromatography but we haven't covered it in lecture yet. Also, this week in lab, is our proticol only covering up to the salting out procedure? thanks!