1. Protein Purification Lab C2
Pages 101 to 142
Lab C.2
Four Periods
Protocol Page 118-142
Be sure to read theory starting
page 104

2. Exam
• Exam March 14
• 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 118
• 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
• Salting IN • Salting OUT
• At low concentrations, • At high concentrations
added salt usually added salt lowers the
increases the solubility of solubility of
charged macromolecules macromolecules because
because the salt screens it competes for the
out charge-charge solvent (H2O) needed to
interactions. solvate the
• So low [salt] prevents macromolecules.
aggregation and therefore • So high [salt] removes
precipitation or the solvation sphere from
“crashing.” the protein molecules and
they come out of solution.

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

13. Dialysis
• 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 125

15. Gel Filtration

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

17. Ion Exchange

18. Affinity Chromatography
We will use bound Adenosine
-5’-monophosphate. This is
part Of NAD+. LDH will
Bind. Release LDH by adding
NADH

19. NAD+
AMP

20. 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 ad NADH which binds tighter to the
LDH.

21. Protein Purification
page 130
Activity
A280
NADH

22. Protein Concentration
• Lowry ( most cited reference in biology)
– Color assay
• A280
– Intrinsic absorbance
– Relies on aromatic amino acids
• BCA page 133 & 137
– Modification of Lowry: increased sensitivity and
consistency
• Bradford
– Shifts Amax of dye from 465nm to 595nm

23. A280 Page 114 &131
• Uses intrinsic absorbance
• Detects aromatic residues
– Resonating bonds
• Depends on protein structure, native state
and AA composition
• Retains protein function

24. PAGE Apparatus (purity and MW)
4th week Page 135, 142

25. Protein separation using SDS-PAGE
(Laemmli system)
1. Apply protein/dye samples 2. Run the electrophoresis until dye
into polyacrylamide gel wells 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
1. Complete mix of proteins
2. High Salt
3. Ion exchange
4. Gel-filtratio
5. 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 119
Ground sirloin
(or alternative LDH source)
Place in blender, add buffer,
homogenize
Initial meat suspension
Centrifuge
Discard
precipitate
(save 1 ml)
Cleared meat Step 1
extract
Ammonium sulfate
precipitation, Centrifuge We will do only one NH4SO4 cut
Supernate
Precipitate
(save 1 ml)
Save 3 samples
Resuspend in
Step 2a buffer Will determine protein concentration
(save 1 ml)
Step 2b activity and purity
Discard
remainder Add PMSF,
Dialyze
Remove dialysate,
Store at -20oC

30. Will fill out this critical table as we
proceed page 138
Table C.2-4. Enzyme Purification Table
Net volume V0 units per V0 units Protein Protein Net Specific
(ml) ml Total content concentration amount Activity
(an “amount”) (% of total) (mg/ml) of protein (V0/mg
(mg) protein)
Step A B C D E F G
1.
Cleared
2.
(NH4)2SO4
Supernata
nt
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 118 (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 122
– 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 124
Blank without NADH Blank with NADH
A B
0.4 0.4
observed 0.2
0.2
observed
0 0
0 60 120 180 0 60 120 180
time (sec) time (sec)
extrapolated
timecourse

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 122)
Prepare the reaction mixtures
Each reaction will contain 3.200 ml: Zero the spectrophotometer:
3.00 ml 50 mM buffer, pH 7.5 Add buffer and pyruvate
to the cuvette then set
50 µl NADH the zero.
50 µl pyruvate Add NADH and check
the A340 value.
100 µl Enzyme solution,
column fraction
or diluted Step 1, 2, 3 or 4
Determine A340 at 15 sec and 45 sec
after adding the enzyme sample.
Note: You may have to adjust the
time frame of the rate measurement
or the amount of added enzyme to
achieve a non-spurious V0 value.
Calculate V0.
Divide the raw answer by the
product of ε340 (for NADH)
times the cuvette path length to
convert the units to mole/liter
per sec units.

37. Spurious Vo Measurements
Same as with ADH
(this is similar to your [ADH] exp)
A) Small [E] B) Increasing [E]
0.6 0.6
0.4 more 0.4
enzyme
A340 A340
0.2 0.2
0 0
0 15 30 45 60 75 0 15 30 45 60
time (sec) time (sec)

38. Procedure (Page 122)
• 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 123
Table C.2-3. Lactate Dehydrogenase Reaction Time Courses
Reading time A340 readings
number (seconds)
50 ml 100 ml 200 ml 300 ml 400 ml sample
sample sample sample sample
1 0
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 138)

43. Will generate this elution profile
Page 130
contaminant protein
LDH
A280 V0
NADH
added
0
0 10 20 30 40 50 60 70 80
fraction (tube) number
(approximate only)

44. Will fill out this critical table as we
proceed page 138 (day 4)
Table C.2-4. Enzyme Purification Table
Net volume V0 units per V0 units Protein Protein Net Specific
(ml) ml Total content concentration amount Activity
(an “amount”) (% of total) (mg/ml) of protein (V0/mg
(mg) protein)
Step A B C D E F G
1.
Cleared
2.
(NH4)2SO4
Supernata
nt
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