Originated by John Tymoczko

Updated by Rebecca Bryant

Mastery of a protein purification scheme is a rite of passage for all biochemists. The techniques employed in protein purification utilize important biochemical ideas about the structure, catalytic reactivity, and other characteristics of the protein of interest.

We will perform a two step purification of LDH from chicken breast muscle. Lactic Acid Dehydrogenase in an important enzyme in the anaerobic metabolism of glucose for the generation of ATP. The activity of this enzyme is primarily responsible for explosive anaerobic athletic activity. The reaction that the enzyme catalyzes is the interconversion of pyruvate and lactate. Under anaerobic conditions, pyruvate is converted to lactate, with the concomitant conversion of NADH to NAD⁺. The regeneration of the NAD⁺ permits continued metabolic flux down the glycolytic pathway. This flow continues until the energy demands cease or until the cell and blood levels of lactate become intolerably high.

Pyruvate + NADH ------------------> Lactic acid + NAD⁺

LDH

LDH is a tetramer of 35 kd subunits. There are 2 types of subunits: and H form which predominates in the heart and an M form that predominates in the muscle and liver. These subunits can associate to form five types of tetramers (H4, H3L1, H2L2, H1L3, L4), all with LDH activity but with different substrate affinities and different responses to allosteric effectors. In general, enzymes that catalyze the same reaction but which differ in structure are referred to as isozymes or isoenzymes. You will appreciate the tertiary structure of LDH after completing Homework I….can you stand the anticipation?

For your enjoyment, your TA has spent countless hours in the preparation of crude chicken breast extract. He should be praised accordingly. Frozen chicken breast cubes (50 gm) are placed in 80 ml of extraction buffer (20mM Tris-HCl, pH 8.6-1mM b -mercaptoethanol (BME)-1mM Phenylmethylsulfonyl Fluoride (PMSF). The cubes are blended at "high" in an ice-cold Waring blender until the chicken is homogenized. The homogenate is placed into a beaker and the blender is washed with 20 ml of extraction buffer. This is combined with the homogenate. This is then stirred at 0-4oC for 1 hr. (What is happening here? Why must it be kept cold?) Take an aliquot of the homogenate and strain it through a double layer of cheese cloth into a 50 ml centrifuge tube on ice. The extract is centrifuged at 8850 rpm in the Sorvall centrifuge and the sup (pronounced "soup" ) is used as the crude extract. Remember, your are working with raw chicken. We are in Salmonella City.

Despite the presence of phenylmethylsulfonyl fluoride (a protease inhibitor) (What kind of protease would this inhibit?), the LDH is very sensitive to proteolysis. The extract must be assayed for enzyme activity today.

Purification Step 1--DEAE-cellulose chromatography

1. Locate, with the help of your instructor and/or lab assistant, a minicolumn.

2. Set up the column in a clamp on a ring stand and attach the tube assembly. Make sure the column is stopped. Add approx. 3 ml of buffer A (20 mM Tris-HCl, pH 8.6, 1 mM BME to the column. You should be using COLD buffer A.

3. Stir or gently swirl the DEAE slurry and transfer 3 ml (packed volume) of the matrix to the column. Wash the column with 20 ml of buffer A. Never let the column dry out. (Why is this especially important?)

4. Remove 300 m l of your crude extract for enzyme assay, determination of protein concentration, and SDS-gel electrophoresis.

5. Load 3 ml of sample onto the column and begin collecting 3 ml fractions. Wash the column with 4 x 3 ml aliquots of Buffer A. Save all fractions for assay. (What can you tell about the net charge on your enzyme from this part of the lab?)

We will determine the LDH activity spectrophotometrically by determining the amount of NAD+ converted to NADH. This takes advantage of the fact that NADH has an absorbance peak at 340nm while NAD+ exhibits little absorbance at this wavelength. We are also, of course, converting lactate into pyruvate, but this conversion is not measured as readily.

1. Determine how many tubes you will assay. Do duplicates and remember blanks. Add 2 to this number as a sacrifice to the gods of enzyme assays.

2. Multiply the number determined in 1 by the following:

1.4 ml of 99 mM lithium lactate in 10 mM Tris-HCl, pH8.6

0.7 ml of 0.7mM NAD+ in 10 mM Tris-HCl, pH 8.6

0.4 ml of 0.5M NaCl in 19 mM NaHCO3.

3. Mix well and pipette 2.5 ml into each plastic disposable cuvette.

4. Add 10 m l of sample to the reaction tube. Cover with parafilm and invert to mix. At exactly 1 min read absorbance at 340 (A340). Make sure to do the blank first to zero your spectrophotometer. Repeat for each fraction. If you get a very low reading for a fraction, you should re-assay with a fresh tube of reaction mix and more sample. Add up to 50 m l of sample. If that doesn't do the trick, cash in your chips as you have no LDH activity.

5. Repeat steps 3 and 4 for each fraction to be assayed.

6. SAVE THE FRACTIONS WITH THE HIGHEST ENZYME ACTIVITY. IMMEDIATELY REMOVE 0.5 ML FOR PROTEIN CONCENTRATION DETERMINATION AND SDS-POLYACRYLAMIDE GEL ELECTROPHORESIS. STORE YOUR MAIN SAMPLE AND THE ALIQUOT THAT YOU JUST REMOVED IN THE FREEZER UNTIL NEXT WEEK. DON’T FORGET TO LABEL THEM WELL!

We will now extend the purification of chicken breast LDH by passing the partially purified DEAE-cellulose eluant over a Cibacron Blue column. This column has the Cibacron blue 3G-A dye attached to agarose, which functions as the inert support. This dye has the shape and charge characteristics that mimic pyridine nucleotides. Consequently, when we pass our DEAE-eluant over the affinity column, only those proteins with an affinity for pyridine nucleotides will bind. The rest can be washed off. Further purification could be obtained by using a second wash that contains NAD+. This would remove proteins with a moderate affinity for the dye. We will skip this step because of general course rules 10 and 11. (Be sure you know those rules.) Finally, LDH (and any other proteins with a high affinity for NADH) will be eluted with buffer containing NADH.

1. Prepare a 0.5 ml column of Cibacron Blue Agarose in one of those spiffy disposable columns.

2. Wash with 10 ml of buffer A (20 mM Tris-HCl, pH 8.6, 1 mM BME).

3. Load your DEAE-cellulose eluant. BE SURE TO SET SOME ASIDE TO DETERMINE PROTEIN CONCENTRATION AND TO RUN ON A GEL (200 m l).

4. Again, collect 3 ml fractions. Wash with 20 ml of buffer A.

5. Wash with 5 ml of NADH buffer (20 mM Tris-HCl, pH 8.6, 1 mM NADH, 0.5 mM BME) and then with 10 ml of buffer A.

7. Assay all fractions for enzyme activity. Determine the protein concentration of your active fraction. Label and store in the freezer.

We will use the Bradford technique for determining protein concentration. This procedure makes use of the dye Coomassie Brilliant blue. When dissolved in acid-alcoholic medium, the dye reacts almost immediately with protein to form a blue colored protein dye complex. The complex causes a shift in the absorption maximum from 465 to 595 nm. The amount of color produced is proportional to protein concentration.

1. Preparation of a standard curve.

50 m l of water only

10 m l of 1 mg/ml albumin + 40 m l of water

25 m l of 1 mg/ml albumin + 25m l of water

50 m l of 1 mg/ml albumin

Do duplicates.

2. To measure the absorbance of your samples you will have to fiddle around with the right dilutions to use. This is important because you need to make sure that all of your absorbency readings are between 0.05 and 1.3. Outside of this range, useful data are not obtained. Here are some dilutions to start with:

crude: 1:100

DEAE fraction: 1:100 or 1:50

Affinity fraction: use straight or 1:5

3. Add 2.5 ml of protein dye to each tube. Mix well. Wait at least 2 min, but not more than 30 min, and read the color at A595. Determine the protein concentration of your sample from the standard curve.

4. Make sure that you have protein concentrations for all appropriate fractions by next lab session. Appropriate fractions include but may not be limited to: Crude extract, DEAE-purified material and Affinity-purified material.

5. Fill out the table on the next page.

A unit will be defined a level of activity that produces 1m mole of NADH per minute at room temperature.

To convert absorbance to concentration, use the following formula (often called Beer’s Law):

Absorbance = (extinction coefficient) x concentration (M) x light path (cm)

0r

A = e cl

where l is almost always 1 cm. and e , which is a characteristic of the molecule in question, is 6220 M^-1 cm^-1 for NADH.

Using Beer’s law, determine the concentration of the NADH. Keep in mind that what you get is a concentration, but your definition of units is m moles formed in 1 min.

Biology is like a totally excellent party

From the protein concentrations that you measured last week, determine the volume of each of your aliquots (Crude Extract, DEAE-fraction and Affinity fraction) that contain 50 m g of protein. Pipette this amount into an appropriately labeled eppendorf tube. Make each of these samples 10% with respect to trichloroacetic acid (TCA) using the 100 % TCA that you are provided. The TCA precipitates the proteins (Why ?) and thus affords us a means of concentrating the sample. If your sample volume is small, as will probably be the case with the Crude Extract, you can make up 10 % TCA and add 1 ml of this directly to the sample. That is, if the sample volume is small, we can ignore its volume and not be concerned that it might dilute the concentration of TCA.

Once all samples are in 10 % TCA, keep on ice for at least 15 min., and then centrifuge 10 min. in the Eppendorf centrifuge in the cold room. Pour off the TCA (remove as much as possible using a pipetteman!! This is trés importante! Rinse the pellet gently with ice-cold acetone. Let air dry for a few minutes). Add 30 m l of 2x protein sample buffer. This consists of Tris-HCl, pH 6.8, sodium dodecyl sulfate (What is this for?), glycerol (Why is this added?) and bromophenol blue (What is the purpose of this?). Your samples should remain blue. If they turn yellow or green, add some 5N NaOH (0.5 to 1.0 m l at a time) until they turn blue again. Vortex. Make sure that your sample is dissolved, i.e. make sure that you can not see the pellet.

Store your samples in the freezer or continue on with the SDS-PAGE.

An important and practical means of purifying and assessing the purity of proteins is via gel electrophoresis. A molecule with a net charge will move in an electric field, a process called electrophoresis. The velocity at which the protein will move in the electrical field is proportional to its charge and inversely proportional to its drag, which is a function of its shape and mass as well as the viscosity of the medium.

Electrophoretic separations are usually carried out in gels. For nucleic acid separations, the gel is most commonly agarose, while for proteins, the gel of choice is polyacrylamide. The acrylamide monomers are linked head to tail, and these chains are then cross-linked by the bis -acrylamide to form a porous gel. The porosity is a function of the degree of cross-linking. Large proteins will move slowly in such a gel, while smaller ones will move more quickly.

Polyacrylamide gel electrophoresis (PAGE) can be used to accurately determine the molecular weights of proteins. Proteins are denatured (converted into linear polymers of amino acids) and then subjected to PAGE. However, if this were all that was done, the PAGE wouldn't yield accurate molecular weights, since the charge on the proteins is likely to vary. For instance, a very large, highly charged protein might move more rapidly (i.e., appear smaller) than a non-charged small protein. This problem was solved with the introduction of the detergent sodium dodecyl sulfate (SDS). SDS not only denatures proteins, it is negatively charged and it binds to all proteins (well, most proteins) at a constant ratio of 1SDS/ 2 amino acids. Thus all proteins will have the same charge to mass ratio, and mobility of the protein in PAGE (actually, now SDS-PAGE) is directly proportional to mass. Proteins differing in mass by only 2% can be distinguished by SDS-PAGE.

Proteins are visualized by any number of staining techniques. Coomassie blue can detect as little at 0.1m g of protein, and silver stain can detect 0.02 m g of protein.

We will provide you with a tube of prestained markers. The "prestain" enables you to see the separation of the markers as the gel runs. Add 1-3 ul of mercaptoethanol to all of the samples, including the standards (What is this the purpose of this?). Please do this in the hood. Boil for 1 min. Cool and load, once you have cast your gel.

1. Locate the gray silicone gasket in the U-shaped groove around the side and bottom of the core unit of the gel rig. Remove the gasket and grease lightly and replace.

2. Lay the core unit on the bench top. Place the aluminum plate against the greased gasket with the notched edges at the top of the unit, near the banana plugs.

3. Lightly grease the spacers and on each side of the plate.

4. Place a clean glass plate on top of the spacers. The tabs on the spines of each spacer should be flush with the outside edges of the plate.

5. Pick up the entire assembly. With one hand, hold it together in an upright position on the bench top. Starting at the top on one side, slide one red spring clamp down to the middle of the side edge. Repeat on the other side. Align the plates and spacers by gently pressing down on the upper edges of the glass and alumina plates as well as on the ends of the spacers.

6. Melt some 1% agarose in running gel buffer. Keep at 60oC.

7. Set the assembly on a glass plate. Using a Pasteur pipette, run a little of the melted agarose along the bottom edge of the sandwich. Capillary action will, in theory, draw the solution up into the sandwich.

8. Prepare the acrylamide solution for the running gel (see below) , and pipette into the gel sandwich up to 1.5 cm form the top.

9. Using the syringe, immediately overlay this gently with some water to maintain a flat surface.

10. Once the running gel has polymerized (approx. 30 min.), remove the water, move the clamps nearer the top of the gel rig, and add stacking gel solution.

11. Insert the comb.

12. Allow to polymerize for 45 min.

13. Gently remove the comb, and move the clamps to a central position , just above the rim of the lower buffer chamber.

14. Place the gel unit in the lower buffer chamber. Position the central core across the center of the buffer chamber and push down gently. The unit should snap into place. Hold the unit by its sides and not by the sandwiches, if you know what is good for you.

15. Fill the sample wells and the upper and lower chambers with Laemmli buffer. Notice that the Laemmli buffer you are provided with is 10X and needs to be diluted to 1X! The upper chamber holds 75 ml and the lower 150 ml..

16. Attach the well location decal to the glass plate of the gel.

17. Load your samples into the wells. What is the order of the samples on your gel? Have you thought about the best order for easiest and most clear interpretation of your results?

18. Place the safety lid on the unit, attach to the power supply, and run the gel at 50 mA constant current per gel. If you have 2 gels running off of one power supply, double the current. It should be complete (tracking dye reaches the bottom of the gel) in about 1 hr.

19. Turn off the power supply.

20. Remove the side clamps and remove he gel sandwich from the core unit.

21. Using an extra spacer, pry open the gel sandwich FROM THE BOTTOM to avoid breaking the ears of the alumina plate. The gel usually sticks to the alumina plate. Place the plate and gel into a Tupperware container with 0.1 % Coomassie Blue staining dye. Leave for 2 hr to overnight.

22. Remove the stain (return to the container). Rinse the gel with destain, and then let the gel sit in destain for several hours to overnight. Be careful. You can destain the proteins, also.

23. Remove the destain (down the drain) and add water. Cover the Tupperware container.

24. Ascertain whether your purification worked.

25. Photograph the results. Prior to proceeding with the silver staining procedure, take a picture of your Coomassie stained gel. Try these settings on the camera.

Fstop: F16

Time: 1/125 sec

26. Proceed with the Silver Stain Procedure (See below).

27. Photograph the results.

28. Compare the results of the Coomassie Blue with that of the Silver Stain.

29. Draw a conclusion about the Staining Procedure.

30. Draw another conclusion about your purification procedure.

PLEASE NOTE: Acrylamide is a potent neurotoxin. Contamination can most easily occur when working with the solid because of the possibility of inhaling polyacrylamide dust. For this reason, premixed solutions can be purchased, and in fact, precast gels can also be purchased. In any case, always wear gloves when working with acrylamide.

After adding all of the components, swirl and pipette into gel sandwich quickly. Polymerization can occur very rapidly!

2x protein sample buffer.

This solution can be used several times.

Dissolve 91 g to Tris base in 300 ml H2O. Adjust pH to 8.8 with HCl. Add H2O to 500 ml total volume. Add 2 gm SDS solid or 20 ml of 10 % SDS. If you do this, remember to add 20 ml less of water. Store in the refrigerator.

Silver staining is a very sensitive method for detecting proteins and nucleic acids in polyacrylamide gels. We will be using a kit produced by the Bio-Rad Company that is 50 fold more sensitive that Coomassie brilliant blue staining for proteins and 2-5 fold more sensitive than ethidium bromide for single and double stranded DNA and RNA

Prior to proceeding with the silver staining procedure, take a picture of your Coomassie stained gel.

Fstop: F16

Time: 1/125 sec

Silver Stain kit components:

Oxidizer -- potassium dichromate and nitric acid

Silver reagent -- silver nitrate

developer -- sodium carbonate and paraformaldehyde

Some important points concerning :

Use only the highest quality water. Contaminants, such as chloride ions, will precipitate silver ions, reducing sensitivity and increasing background.

Gently shake the gels at all steps on the orbital shakers.

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Make sure that all chemicals are at room temperature before using

Timing of the steps is critical, but varies with the gel. There is no stopping point once you start.

Bands usually appear dark brown against a pale background. The duration of the development is approximate, and development must be monitored carefully. Stop development when the bands reach the desired intensity in relation to the background.

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With Type 667 film, F stop should be F32 and speed should be 1/125 sec.

This week we will purify lactate dehydrogenase using the technique of immunopurification. The antibody is a commercial preparation to rabbit muscle LDH (see the data sheet). Some of you will isolate LDH from rabbit muscle and others will test the reactivity of the antibody to chicken breast muscle LDH. We will start off with fresh preparations of both the rabbit and the chicken muscle.

The muscle will be homogenized in Extraction Buffer, as previously described. The preparation will be stirred in the cold room for an hour and centrifuged at 10K rpm for 10 min. To further clarify the preparation, 1 ml aliquots will be centrifuged in an Eppendorf centrifuge for 30 min. This will be the material that you use as your source of LDH.

1. To the eppendorf tube containing 20 m g of the antibody, add 400 m l of the centrifuged crude extract. The antibody that we are using is a polyclonal antibody. Make sure that you understand the difference between a polyclonal and monoclonal antibody.

2. Keep on ice for at least 1 hr with occasional agitation.

3. Add 10 mg of protein G-sepharose. The antibody, and with it the LDH, should bind to the protein G. Mix gently but well. Keep on ice for at least 1 hr. with occasional gentle agitation. Do not mix so vigorously that the protein G sepharose gets splashed onto the sides of the tube where it is liable to stick, forever and ever. That would be bad.

4. Wash the pellet with 1 ml of 0.1% triton X-100 in 0.14 M NaCl-10 mMTris-HCl, pH 8.0 (Triton TS). Centrifuge for 10 sec. and discard sup. (Why do we use triton X-100 instead of some other detergent?)

5. Repeat 2 more times.

6. Wash with 1 ml of TS solution.

7. Wash with 1 ml of 0.05 M Tris-HCl, pH 6.8.

8. Extract sample with 20 m l protein sample buffer without b -mercaptoethanol (BME). Cap the tube securely and heat at 100^oC for 5 min. The BME is omitted to minimize release of the antibody chains themselves from the protein G-sepharose. (Why does this minimize release of antibody chains?) (Why is this a good thing to minimize?)

9. Centrifuge 10 sec and carefully remove the supernatant. This is your sample. Analyze by gel electrophoresis. Remember to add 1-3 m l of BME and re-boil the sample just prior to electrophoresis

10. Perform SDS-PAGE as previously described. Stain with Coomassie blue. Photograph the gel and then silver stain. Photograph the gel

11. Be prepared to explain your results and formulate a plan of attack if the results are less that stellar.