Protein Purification Lab C2Pages 101 to 142
Lab C.2
Four Periods
Protocol Page 118-142
Be sure to read theory starting page 104
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
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.
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
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
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
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
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
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
Ammonium Sulfate pptpage 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”
Salting in / Salting out
• 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.
Kosmotrope vs. Chaotrope
• 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.
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.
Column Chromatography2nd Day Page 125
Gel Filtration
Principles of gel filtration (molecular sieving)
106 Da3x105 Da105 Da104 Da
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
3. High molecular weight macromolecules (higher Stoke’s radius) elute first
4. Determine proteins in eluate using suitable assay
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:
Kav = Ve -VoVt - Vo
Ve – elution volumeVo – void volumeVt – total volume
Kav
Log M.Wt.
Ion Exchange
Affinity Chromatography
We will use bound Adenosine-5’-monophosphate. This is part Of NAD+. LDH will Bind. Release LDH by adding NADH
NAD+
AMP
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.
Protein Purificationpage 130
A280
Activity
NADH
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
A280 Page 114 &131
• Uses intrinsic absorbance
• Detects aromatic residues – Resonating bonds
• Depends on protein structure, native state and AA composition
• Retains protein function
Protein separation using SDS-PAGE(Laemmli system)
Stackinggel
Resolvinggel
1. Apply protein/dye samples into polyacrylamide gel wells
2. Run the electrophoresis until dye reaches the end of the gel
3. Remove the gel from the apparatus and stain for proteins
SDS PAGE of Purification
1. Complete mix of proteins2. High Salt3. Ion exchange4. Gel-filtratio5. Affinity
10micrograms loaded in each lane
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
Will fill out this critical table as we proceed page 138
Table C.2-4. Enzyme Purification Table
Net volume(ml)
V0 units per
ml
V0 units
Total(an “amount”)
Protein content
(% of total)
Proteinconcentration
(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 EColumn D = Column C/first value in Column C
Very Important: Page 124
0
0.2
0.4
time (sec)60 120 1800
0
0.2
0.4
time (sec)60 120 1800
A B
observed
observed
extrapolated timecourse
Blank without NADH Blank with NADH
Spurious Vo MeasurementsSame as with ADH
(this is similar to your [ADH] exp)
0
0.2
0.4
0.6
0 15 30 45 60
more enzyme
time (sec)
A340
0
0.2
0.4
0.6
0 15 30 45 60
time (sec)
A340
A) Small [E] B) Increasing [E]
75
C2-3. Page 123Table 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 0
2 15
3 30
4 45
5 60
6 75
7 90
8 105
9 120
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
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.
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)
Will generate this elution profilePage 130
A280 V0
fraction (tube) number (approximate only)
NADH added
0
0 10 20 30 40 50 60 70 80
contaminant protein
LDH
Will fill out this critical table as we proceed page 138 (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)
Proteinconcentration
(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 EColumn D = Column C/first value in Column C
This Lab
• 4 lab periods
• Prelab= 12 points
• Lab Report= 50 points
• First exam in period 4