Protein Purification Lab C2 Pages 101 to 142 Lab C.2 Four Periods Protocol Page 118-142Be 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 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”
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.
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.
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.
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.
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 Purification page 130 ActivityA280 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) 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
SDS PAGE of Purification1. Complete mix of proteins2. High Salt3. Ion exchange4. Gel-filtratio5. Affinity10micrograms 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
Day 1: See Table C2-2(page 117), Page 118- 124. & 138
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 inStep 2a buffer Will determine protein concentration (save 1 ml) Step 2b activity and purity Discard remainder Add PMSF, Dialyze Remove dialysate, Store at -20oC
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
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
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
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
Very Important: Page 124 Blank without NADH Blank with NADHA 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
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.
Flow chart 1B (page 122) Prepare the reaction mixturesEach 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.
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 enzymeA340 A340 0.2 0.2 0 0 0 15 30 45 60 75 0 15 30 45 60 time (sec) time (sec)
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.
C2-3. Page 123Table 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
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 profile Page 130 contaminant protein LDHA280 V0 NADH added 0 0 10 20 30 40 50 60 70 80 fraction (tube) number (approximate only)
Will fill out this critical table as weproceed 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
This Lab• 4 lab periods• Prelab= 12 points• Lab Report= 50 points• First exam in period 4