Name:____________________________________

PROJECT DESCRIPTION: Complete and Appropriate (5 points) __________

MATERIALS AND METHODS: Complete and Clearly written (10 points): Organized into sections

RESULTS: 20 points

Sample calculations present and accurate. ______________

Protein concentrations of each purification step present and accurate. __________

Enzyme activity of each purification step present and accurate: ____________

Table summarizing purification so far is present and accurate _____________

Explanatory text included, complete and accurate. __________

PLAN FOR COMPLETION: (5 points) present and clearly stated.

UNDERSTANDING of the experiment or procedures is demonstrated in the report. (5 points) ___________

FORM AND READABILITY: ________(5 points) Complete sentences and correct spelling are used throughout the report. The sentences make sense and are clearly worded and easy to read. The report has been proofread.

Notes: Calculate all purification factors relative to the crude extract.

Write the materials and methods section as if your reader were a knowledgeable lab technician.

No need to say that you turned on the spec and set the wavelength at 595. Assumed that the person knows to use a spec. Dialyzer removed from plastic wrap.

Project description should not contain experimental details. It should say generally how the purification will be accomplished - a series of steps, including AS, dialysis and ion exchange chromatography.

Multiple Choice: Circle the correct answer. (2 pts each)

1. In protein purification, dialysis is useful for the following purpose
   1. separation of proteins based on hydrophobicity
   2. salting out of proteins
   3. desalting of protein containing solutions
   4. to exchange buffer solutions that the protein sample is dissolved in
   5. both c and d above.

   
   

2. During purification of an enzyme, specific activity should (increase, decrease) as purification proceeds.

   
   

3. The total amount of protein in your sample based on the BioRad protein assay should (increase, decrease) as purification proceeds.

   
   

4. Which of the following methods are used during the purification of -galactosidase to maintain protein stability.
   1. including dithiothreitol in purification buffers
   2. keeping all protein containing solutions on ice or in the refrigerator oat all times
   3. adding stop solution to the enzyme assays
   4. maintaining the protein and its buffers at pH 4.0.
   5. both a and b above.

   
   

5. Which of the following is the natural substrate of the -galactosidase in E. Coli (a) sidase (b) fructose (c) lactose d) o-nitrophenol (ONP) e) ONPG.

   
   

6. The isoelectric point (pl) of a protein is a) the pH at which there is no net charge on the protein b) the pH at which the protein will bind to an ion exchange column c) the pH at which the protein is the most soluble d) none of the above.

   
   True/False. (2 points each) Circle T or F.

7. It is a reasonable goal to retain all the enzyme activity that you started with during purification of that enzyme.
         T        F

   
   

8. Protein separation by ammonium sulfate precipitation is based on the fact that the solubility properties of proteins are different.
         T        F

   
   

9. (4 points) Short answer: In the space provided next to the protein property below, write out a protein separation technique which is based on that property.
   size (molecular weight):
   ___________________________________________________

   charge (pl):
   ___________________________________________________

   heat stability:
   ___________________________________________________

   solubility:
   ___________________________________________________

   
   

10. (3 points) This question relates to the computer purification exercise. During the course of your protein purification, you decide to run 2-D gel electrophoresis with immunoblotting to determine the purity of your sample. The following result is obtained. The protein which shows up on immunoblot is circled with dotted line. Based on the information given, the best method to remove the two contaminating proteins is probably which one of the following methods: (Assume that the stability range of your protein is not an issue)

    

    1. gel filtration chromatography
    2. ion exchange chromatography
    3. heat denaturation
    4. none of the above.

    
    

11. Fill in the Blanks: (5 points)

    In the space provided, write out the best answer for the unit of measurement for each of the terms listed below. For some terms, there may be more than one correct answer.

    volume:
    ___________________________________________________

    concentration:
    ___________________________________________________

    weight:
    ___________________________________________________

    specific activity:
    ___________________________________________________

    units of enzyme activity:
    ___________________________________________________

    microgram (µg)	mg/ml
    milliliter (ml)	nanomoles of product formed perminute at 37 oC
    milligram (mg)	molarity

    

    
    

12. (4 points) If your stock solution is 0.5 mg/ml, how much of your stock do you need to make 1 ml of a 5µg/ml solution.

    
    

13. (5 points) You would like to test your protein extract for enzyme activity but you didn't want to use more than 10 µl of your sample because you want to conserve as much of your enzyme as possible. You decide to test three different dilutions, 1/100, 1/500 and 1/1000. You will need 20 µl of each dilution in order to perform the enzyme assay. Explain how you would make the dilutions accurately. Assume it is not accurate to measure less than 5 µl.

    
    

14. (3 points) You are attempting to follow a laboratory protocol. It says to centrifuge the preparation for 10 minutes at 10,000 rpm in the SE-12 rotor. Because of the size of your samples, you need to use the GSA rotor instead of the SE-12. How would you modify the protocol for your samples. Use the table.

    
    

15. (10 points total) In the process of your -galactosidase protein purification, you want to determine the specific activity of your extract. You decide to perform the BioRad protein microassay protocol just as written in your lab manual. You dilute a sample which contains your protein 1/20 in order to perform protein and enzyme assays. You take 20 µl of the diluted sample and add 780 µl of water just as it states in your lab manual protocol. You then add 200 µl of the Coumassie blue dye and measure the absorbance as directed.

    Absorbance of protein standards
    µg protein of standard	Absorbance at 595nm
    0.8	0.055
    4.0	0.241
    8.0	0.442
    16.0	0.846
    20.0	0.993

    Absorbance at 595nm of diluted extract (1/20 dilution) = 0.632

    1. Use the graph paper provided to make your standard curve.

    2. What is the concentration of total protein in the diluted sample in µg/ml.

    3. What is the concentration of total protein in the original sample in mgs/ml.

    4. If your original sample had a total volume of 5 ml, what is the total amount (weight) of protein you have.

    5. If the enzyme activity of the undiluted sample is 500,000 units/ml, what is the specific activity of your -galactosidase?

    
    

16. BONUS: (3 points) Why does 1M Na₂HCO₃ stop the activity of -galactosidase?

1. You have been given a project to improve the purification protocol for an enzyme, MATCase, which has great potential benefit for the agricultural industry in Wisconsin. Toward this goal, your first assignment is to determine the specific activity of a partially purified enzyme fraction from a previous purification attempt. You go to the -70 freezer, box labeled "MATCase purification", and remove an aliquot labeled "semi-purified MATCase, approximately 0.35 mgs/ml". Your supervisor has indicated that you should not be satisfied with any labeling left by the previous person and wants you to repeat the protein determination.

   a) 20 points. Outline the steps you would need to do in order to perform the BIORAD protein microassay procedure and determine the protein concentration of your enzyme sample. Assume that the solvent is water and that you can use bovine serum albumin as the standard protein (comes from Sigma as a powder). How would you make the stock standard solution, set up standard dilutions for standard curve, make dilutions of the enzyme fraction, etc.

   Tube #	Conc. of Std	Vol. of Stock	Vol. of H20	µg assayed
   1
   2
   3
   4
   5

   b) 20 points. You did the specific activity determination on the previous MATCase preparation and it was worse than the crude extract and even worse than your supervisor had feared. She now wants you to design a purification scheme for this enzyme. You spend some time asking questions about the previous work and list additional characteristics of the enzyme:

   pH stability -- 6.5-9.5
   pl -- not known
   temperature stability -- stable to 45 ^oC for short periods (ten minutes) of time
   Maintains good activity in TRIS buffers.
   Enzyme assay -- enzyme kinetics well understood by biochemistry people with company; assay works well.
   

   Outline a strategy to purify this enzyme. (There isn't only one answer- many possible good answers) How would you go about finding properties and behaviors of this protein which would help you?

2. 5 points. Explain why it is most accurate to do a standard curve each time you want to determine the value of an unknown when using a colorimetric assay (dye based) like the BIO-RAD.

3. 5 points. If you perform the Bio-RAD protein assay and one of your unknowns is too concentrated to read in your standard curve, would it be appropriate to dilute that sample so that it will like within the linear range? Yes or No. Explain why or why not.

4. 10 points. It's Friday afternoon and someone used the last of the enzyme assay buffer. Life is hard but you have to make some more. One of the components in the recipe is 0.5 gms of MgCl₂. When you go to the shelf, you find only MgCl₂^.6 H₂0. How would you make the substitution? Show all work. Circle answer.

5. 5 points. How many grams of NaCl are in 250 µl of 2.5 mM NaCl.

6. 5 points. How many millimoles of NaCl are there in 10 mls of a 45% solution?

7. 5 points. If you make a 1/1000 dilution of your -galactosidase in order to assay the enzyme activity and it turns bright yellow immediately after adding the ONPG, would this assay be accurate? Explain your answer. What would you do about it. How would you choose the dilution factors and how would you make this dilution or dilutions. Assume that you want to use as little of your precious enzyme sample as possible.

8. If you need to make some NTM buffer with 0.3 M NaCl, can you dilute NTM buffer with 0.4M NaCl with water in order to make NTM with 0.2. Explain why or why not?
   Can you make 0.3 M NaCl

   Can you make 0.32 M NaCl. Explain your reasoning.

9. Explain why ammonium sulfate is most often used for salting out procedures.

10. You have just been given a lab project to modify the enzyme assay protocol for measuring the activity of -galactosidase using ONPG as substrate. The volumes must be adjusted for a new spectrophotometer with very small cuvettes.
    How will you modify this assay so that the total assay volume is 0.5 mls.

    How would you re-design the assay if you want to make sure that the enzyme has the same number of moles of substrate available as in the larger volume assay. Show all work.

    What the conversion factor be 380 or would it change?

    What would it be? Explain your reasoning?

    What is the wavelength for enzyme assay? Why?

    What is the wavelength of the Biorad assay? Why?

    Why do we measure the Absorbance at 280 for our fractions off the ion-exchange column?

    How does Na₂CO₃ function as a stop solution for the -galactosidase assay?

11. 5 points. During -galactosidase purification, what if you made a mistake during the -galactosidase purification and used 0.4 M NTM buffer instead of 0.2 M NTM buffer to dialyze your sample. What would be the result of this mistake when loading your dialysate on the DEAE ion-exchange column? What could you do to correct this mistake?

12. 20 points. The following table documents the results of a protein purification procedure.

    a) Fill in the table below:

    Purification Step	Enzyme Units/ml	Protein mg/ml	Volume of fraction	Units in total volume	Yield %	Specific Activity	Purification Factor
    Crude	100,000.00	20.0	40 ml		100		1
    Ammonium Sulfate pellet - resuspended	100,000.00	10.0	10 ml
    Dialysate	70,000.00	3.5	12 ml
    Ion Exchange	7,000.00	0.35	4 ml

    b) Discuss the results of this purification? How successful was this purification attempt? Did specific activity and yield change in the expected manner at each step? What modifications would you make in your purification process next time based on the results in this table?

13. 10 points. The following questions concern analysis of yield during enzyme purification.
    1. If you have 0.5 ml of an enzyme solution containing 200 units/ml, how many units do you have?

    2. If you have 10mls of a solution containing 200 units/ml, how many units do you have?

    3. When comparing the percent yield from one purification step to the next (say between crude extract and ammonium sulfate pellet), is it useful to express the enzyme activity in units/ml if the volumes are not equal. Explain. Why or why not? If answer is no, what is an alternative way to express the units in order to compare the yield.

14. 10 points. You would like to solve the world's garbage and landfill problems and maybe make some dollars for yourself along the way. After years of research in the field, you have isolated very small amounts of an enzyme, garbagase, that degrades a type of plastic that is used in many throwaway containers (as well as the plastic in trashbags and disposable diapers). You have been able to produce a reliable antibody and work out the conditions for a reliable enzyme assay. However, this enzyme is only found in very small quantities, produced by a rare species of fungi only when in the presence of a really foul and toxic (to humans) compound. This compound apparently induces the expression of this enzyme. You decide to clone this enzyme in E. Coli so that it is produced consitutively (doesn't need toxic compound for expression). After many attempts, you succeed in cloning the enzyme. The recombinant enzyme has the same molecular weight as fungal enzyme and is recognized by the antibody to fungal garbagase.

15. 20 points. You want to determine the amount of total protein in your 50 mls of crude extract. You set up standards for the BioRad microassay. You dilute an aliquot of the crude extract 1:10. You then remove 20 µl of the diluted crude extract for assay, adding 780 µl of H₂0 and then 0.2 of Bio Rad dye reagent. 15 minutes later, you read the OD at 595 nm and determine that there are 2 µg in the tube that you assayed.
    1. What is the protein concentration of the original sample in mg/ml.

    2. What is the specific activity of the crude extract if the original sample has 15,000 units/ml.

    3. How much protein do you have in the entire volume of crude extract.

16. 20 points.
    1. If you have 100 mls of an enzyme preparation and the amount of enzyme activity in the preparation is 1,000 units per ml, how many milligrams of enzyme do you have in the total volume of crude extract if the specific activity of purified enzyme is 2,000 units/mg.

    2. Suppose that your goal for this purification step was to obtain at least 0.5 gm of enzyme. Do you have enough