Protein Purification Lab C2 Pages 115 to 168

1. Protein Purification Lab C2Pages 115 to 168 Lab C.2 Four Periods Protocol Page 135-160 Benchtop Protocols begin on page 398 Be sure to read theory starting page 120

2. Exam • Exam March 3,4 • Includes Carbohydrates, Enzyme kinetics, and all protein labs and material related there to. • Pay attention to the powerpoints • Read theory sections in the lab manual • Will be about one hour in length • Example of exam with answers is posted on web

3. You Have: • Become skilled at using micro pipetters • Have learned to use the spectrophotometer • To determine concentration of an unknown • Beers Law • To measure activity of an enzyme • Have learned how to organize experimental protocols • Have learned how to prepare a report.

4. In the next days • You will use all of these skills to perform a fundamental exercise in Biochemistry/Molecular Biology • Will learn basic protocols in protein purification and analysis

5. Protein Purification • A black art (proteins have personality) • Requires knowledge of protein • What kind of cell is it coming from • What part of cell • What does it do • Particularly helpful • Size • Composition

6. Strategy • Move from organism to pure protein in as few steps as possible with as little loss of activity (assayable quality) as possible • Time and temperature are factors

7. Protocols for Protein Purification • Highly individualized • Use a common approach • Fractionate crude extract in a way that protein of interest always goes into the pellet or the supernatant. • Follow progress with functional assay

8. Lactate Dehydrogenase • NADH + H+ + Pyruvate =NAD+ + Lactate • Enzyme clears lactic acid from working muscles • The obvious source of enzyme is muscle tissue (heart & skeletal muscle, H&M, isomers) • We will assay for the enzymes ability to convert Pyruvate to Lactate

9. Begin with intact tissue • Disrupt (step4&5) • Blender, homoginizer • Remove debris (step7) • Centrifugation • Precipitate/concentrate (step 14-16) • Ammonium sulfate • Remove salt (step 22) • dialysis • Purify (next Lab) • Chromatography • Analyze (Part B and week 3 & 4) • Activity, molecular weight

10. Ammonium Sulfate pptpage 124 • Has a wide range of application • Relies on fact that proteins loose solubility as concentration of salt is increased • Is characteristic of particular protein • Results in a partial purification of all proteins with similar solubility characteristics • Must determine [amm sulf] to precipitate your protein empirically. • Produces “salt cuts”

11. Salting IN At low concentrations, added salt usually increases the solubility of charged macromolecules because the salt screens out charge-charge interactions. So low [salt] prevents aggregation and therefore precipitation or “crashing.” Salting OUT At high concentrations added salt lowers the solubility of macromolecules because it competes for the solvent (H2O) needed to solvate the macromolecules. So high [salt] removes the solvation sphere from the protein molecules and they come out of solution. Salting in / Salting out

12. Ammonium Sulfate Increasing conc causes proteins to precipitate stably. Kosmotropic ion = stabilizing ion. Urea Increasing conc denatures proteins; when they finally do precipitate, it is random and aggregated. Chaotropic ion = denaturing ion. Kosmotrope vs. ChaotropePage 125

13. Dialysis Page 180 • Passage of solutes through a semi-permeable membrane. • Pores in the dialysis membrane are of a certain size. • Protein stays in; water, salts, protein fragments, and other molecules smaller than the pore size pass through.

14. Column Chromatography2nd Day Page 126

15. Available in any volume

16. Gel Filtration

17. Principles of gel filtration (molecular sieving) 5. Estimate approximate molecular weight of unknown proteins and/or protein complexes using calibration curve with pre-run standard proteins of known M.Wt. and the following formula: 2. Collect fractions, typically 120 from a 1.5x100 cm column. Do not change buffer composition 1. Apply a mixture of proteins on a gel filtration column (Sepharose, Sephacryl, etc) 3. High molecular weight macromolecules (higher Stoke’s radius) elute first Ve -Vo Vt - Vo Ve – elution volumeVo – void volumeVt – total volume Kav = 106 Da 3x105 Da 105 Da 104 Da 4. Determine proteins in eluate using suitable assay Kav Log M.Wt.

18. Ion Exchange

19. Affinity Chromatography We will use bound Adenosine -5’-monophosphate (page 128 & 152). This is part Of NAD+. LDH will Bind. Release LDH by adding NADH

20. NAD+ AMP

21. Affinity chromatography • Remember: NADH is a co-substrate for lactate dehydrogenase. • We use AMP-Sepharose: AMP is covalently bound to the affinity gel, which will not pass through the filter. • LDH binds to the AMP b/c it looks like half an NADH. • Thus LDH remains immobilized in the column until we add NADH which binds tighter to the LDH.

22. Protein Purificationpage 130 Activity A280 NADH

23. Protein Concentration Lowry ( most cited reference in biology) Color assay A280 Intrinsic absorbance Page 132 Relies on aromatic amino acids BCA page 133 Modification of Lowry: increased sensitivity and consistency Bradford Shifts Amax of dye from 465nm to 595nm

24. A280 Page 132 • Uses intrinsic absorbance • Detects aromatic residues • Resonating bonds • Depends on protein structure, native state and AA composition • Retains protein function

25. Protein separation using SDS-PAGE(Laemmli system) Page 158 2. Run the electrophoresis until dye reaches the end of the gel 1. Apply protein/dye samples into polyacrylamide gel wells Stackinggel Resolvinggel 3. Remove the gel from the apparatus and stain for proteins

26. SDS PAGE of Purification Process • Complete mix of proteins • High Salt • Ion exchange • Gel-filtratio • Affinity 10micrograms loaded in each lane

27. IMPORTANT • Do not throw away anything until you are certain you no longer need it • Biggest source of problem in this lab • Label everything clearly copy labels into lab book • Throwing out wrong fraction results in starting over • 3 days into experiment huge problem

28. Will follow Flow sheet: Page 138 We will do only one NH4SO4 cut Save 3 samples Will determine protein concentration activity and purity

29. Will fill out this critical table as we proceed page 162 Column C = (Column A)(Column B) Column F = (Column A)(Column E) Column G = Column C/Column F = Column B / Column E Column D = Column C/first value in Column C

30. Today. Page 138 (part of group) • Steps 1-5: Weigh muscle sample place in blender with 50ml ice cold buffer homogenize for 2 minutes. • Steps 6&7: remove large debris by centrifugation Save Supernatant (remove 1ml (Microfuge tube) for later analysis). • Steps 9-13: Measure the volume of the supernatant determine amount of ammonium sulfate required for precipitation, weigh out 0.4 grams per/ml (NH4)2SO4

31. Today group 1 continued • Step14-16: Slowly add salt to gently stirred supernatant . Keep Cold!!See step 12 • Step 17: Centrifuge precipitate to a pellet • Step 18-21: Save supernatant (1ml in microfuge tube). Suspend pellets in 5ml cold buffer • Step 22, 23: Add PMSF and place suspended pellet in dialysis tubing and give to TA

32. Today group 2 • Set up standard assay as on page 142 • Measure loss of absorbance as NADH is converted to NAD+ • Step 4 is similar to Kinetic curve you did for ADH (page 124) only reversed as measure loss of absorbance • Steps 8-12: You will determine the velocity of LDH catalyzed reaction by varying the concentration of LDH with constant substrate and cofactor. Be sure to adjust the amount of reaction buffer to give 3.2 ml final volume in each assay

33. Very Important: Page 145 Blank without NADH Blank with NADH

34. Today group 2 continued • You are establishing the assay conditions you will use next week to follow the purification of LDH. You must become proficient at this assay.

35. Flow chart 1B (page 142)

36. Spurious Vo MeasurementsSame as with ADH(this is similar to your [ADH] exp)

37. Procedure (Page 143) • 1 Step 1-6. Will create a kinetic curve for LDH (adjust volume of buffer to make 3.2ml) • Similar to ADH • 2. Repeat kinetic curve with different concentrations of enzyme • This is protocol you will use as you purify LDH • Do this assay on the unknown samples from step one and 2a from group 1.

38. C2-3. Page 144

39. Next Week Column Chromatography • Due next time: Prelab assignment for period 2 of ‘LDH Purification’ • You really should write up or otherwise arrange what you did today as soon as possible. Do Not Trust Your Memory

40. Next lab • Need member of group to be here at 1:30 to begin washing column • Will need to measure absorbance at 280 to determine that contaminating protein is lost from column. Wash and measure until A280 is constant.

41. Strategy • For samples generated determine amount of protein (A280 ) and activity • Activity per microgram of protein =s specific activity • You strive for maximal activity per unit of protein. (table C2-4 Column G, Page 162)

42. Will generate this elution profilePage 153

43. Will fill out this critical table as we proceed page 162 (day 4) Column C = (Column A)(Column B) Column F = (Column A)(Column E) Column G = Column C/Column F = Column B / Column E Column D = Column C/first value in Column C

44. This Lab • 4 lab periods • Prelab= 12 points • Lab Report= 50 points • First exam in period 4