1. Protein Purification Lab C2 Pages 115 to 168
Four Periods
Protocol Page
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 ppt page 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 / Salting out
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.

12. Kosmotrope vs. Chaotrope Page 125
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.

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 Chromatography 2nd Day Page 126

15. Available in any volume

16. Gel Filtration

17. Principles of gel filtration (molecular sieving)
1. Apply a mixture of proteins on a gel filtration column (Sepharose, Sephacryl, etc)
2. Collect fractions,
typically 120 from a 1.5x100 cm column. Do not change buffer composition
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:
3. High molecular weight macromolecules (higher Stoke’s radius) elute first
Ve -Vo
Vt - Vo
Ve – elution volume Vo – void volume Vt – total volume
Kav = Da
3x105 Da Da 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 Purification page 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)
1. Apply protein/dye samples into polyacrylamide gel wells
2. Run the electrophoresis until dye reaches the end of the gel
Stacking gel
Resolving gel
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. Day 1: See Table C2-2 (page 117), Page 118-124. & 138

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

30. Will fill out this critical table as we proceed page 162
Table C.2-4. Enzyme Purification Table
Net volume
(ml)
V0 units per ml
V0 units
Total
(an “amount”)
Protein content
(% of total)
Protein
concentration
(mg/ml)
Net amount
of protein
(mg)
Specific Activity
(V0/mg protein)
Step A B C D E F G
1. Cleared
2. (NH4)2SO4 Supernatant
3. diluted dialyzed sample/ solution placed on column
4. pooled peak tubes from column
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

31. 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

32. 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

33. 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

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

35. 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.

36. Flow chart 1B (page 142)

37. Spurious Vo Measurements Same as with ADH (this is similar to your [ADH] exp)

38. 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.

39. C2-3. Page 144
Table C.2-3. Lactate Dehydrogenase Reaction Time Courses
Reading number
time
(seconds)
A340 readings
50 ml
sample
100 ml sample
200 ml sample
300 ml sample
400 ml sample 1 2 15 3 30 4 45 5 60 6 75 7 90 8
105 9
120

40. 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

41. 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.

42. 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)

43. Will generate this elution profile Page 153

44. Will fill out this critical table as we proceed page 162 (day 4)
Table C.2-4. Enzyme Purification Table
Net volume
(ml)
V0 units per ml
V0 units
Total
(an “amount”)
Protein content
(% of total)
Protein
concentration
(mg/ml)
Net amount
of protein
(mg)
Specific Activity
(V0/mg protein)
Step A B C D E F G
1. Cleared
2. (NH4)2SO4 Supernatant
3. diluted dialyzed sample/ solution placed on column
4. pooled peak tubes from column
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

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