Presentation on theme: "Protein Purification Lab C2 Pages 115 to 168"— Presentation transcript:
1Protein Purification Lab C2 Pages 115 to 168 Four PeriodsProtocol PageBenchtop Protocols begin on page 398Be sure to read theory starting page 120
2ExamExam March 3,4Includes Carbohydrates, Enzyme kinetics, and all protein labs and material related there to.Pay attention to the powerpointsRead theory sections in the lab manualWill be about one hour in lengthExample of exam with answers is posted on web
3You Have: Become skilled at using micro pipetters Have learned to use the spectrophotometerTo determine concentration of an unknownBeers LawTo measure activity of an enzymeHave learned how to organize experimental protocolsHave learned how to prepare a report.
4In the next daysYou will use all of these skills to perform a fundamental exercise in Biochemistry/Molecular BiologyWill learn basic protocols in protein purification and analysis
5Protein Purification A black art (proteins have personality) Requires knowledge of proteinWhat kind of cell is it coming fromWhat part of cellWhat does it doParticularly helpfulSizeComposition
6StrategyMove from organism to pure protein in as few steps as possible with as little loss of activity (assayable quality) as possibleTime and temperature are factors
7Protocols for Protein Purification Highly individualizedUse a common approachFractionate crude extract in a way that protein of interest always goes into the pellet or the supernatant.Follow progress with functional assay
8Lactate Dehydrogenase NADH + H+ + Pyruvate =NAD+ + LactateEnzyme clears lactic acid from working musclesThe 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
9Begin with intact tissue Disrupt (step4&5)Blender, homoginizerRemove debris (step7)CentrifugationPrecipitate/concentrate (step 14-16)Ammonium sulfateRemove salt (step 22)dialysisPurify (next Lab)ChromatographyAnalyze (Part B and week 3 & 4)Activity, molecular weight
10Ammonium Sulfate ppt page 124 Has a wide range of applicationRelies on fact that proteins loose solubility as concentration of salt is increasedIs characteristic of particular proteinResults in a partial purification of all proteins with similar solubility characteristicsMust determine [amm sulf] to precipitate your protein empirically.Produces “salt cuts”
11Salting 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 OUTAt 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.
12Kosmotrope vs. Chaotrope Page 125 Ammonium SulfateIncreasing conc causes proteins to precipitate stably.Kosmotropic ion = stabilizing ion.UreaIncreasing conc denatures proteins; when they finally do precipitate, it is random and aggregated.Chaotropic ion = denaturing ion.
13Dialysis Page 180Passage 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.
17Principles 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 composition5. 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 firstVe -VoVt - VoVe – elution volume Vo – void volume Vt – total volumeKav =Da3x105 DaDaDa4. Determine proteins in eluate using suitable assayKavLog M.Wt.
21Affinity 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.
23Protein Concentration Lowry ( most cited reference in biology)Color assayA280Intrinsic absorbance Page 132Relies on aromatic amino acidsBCA page 133Modification of Lowry: increased sensitivity and consistencyBradfordShifts Amax of dye from 465nm to 595nm
24A280 Page 132 Uses intrinsic absorbance Detects aromatic residues Resonating bondsDepends on protein structure, native state and AA compositionRetains protein function
25Protein separation using SDS-PAGE (Laemmli system) 1. Apply protein/dye samples into polyacrylamide gel wells2. Run the electrophoresis until dye reaches the end of the gelStacking gelResolving gel3. Remove the gel from the apparatus and stain for proteins
26SDS PAGE of Purification Process Complete mix of proteinsHigh SaltIon exchangeGel-filtratioAffinity10micrograms loaded in each lane
27IMPORTANTDo not throw away anything until you are certain you no longer need itBiggest source of problem in this labLabel everything clearly copy labels into lab bookThrowing out wrong fraction results in starting over3 days into experiment huge problem
29Will follow Flow sheet: Page 138 We will do only one NH4SO4 cutSave 3 samplesWill determine protein concentrationactivity and purity
30Will fill out this critical table as we proceed page 162 Table C.2-4. Enzyme Purification TableNet volume(ml)V0 units per mlV0 unitsTotal(an “amount”)Protein content(% of total)Proteinconcentration(mg/ml)Net amountof protein(mg)Specific Activity(V0/mg protein)StepABCDEFG1. Cleared2. (NH4)2SO4 Supernatant3. diluted dialyzed sample/ solution placed on column4. pooled peak tubes from columnColumn 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
31Today. 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
32Today group 1 continuedStep14-16: Slowly add salt to gently stirred supernatant . Keep Cold!!See step 12Step 17: Centrifuge precipitate to a pelletStep 18-21: Save supernatant (1ml in microfuge tube). Suspend pellets in 5ml cold bufferStep 22, 23: Add PMSF and place suspended pellet in dialysis tubing and give to TA
33Today 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 absorbanceSteps 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
34Very Important: Page 145Blank without NADH Blank with NADH
35Today group 2 continuedYou are establishing the assay conditions you will use next week to follow the purification of LDH. You must become proficient at this assay.
37Spurious Vo Measurements Same as with ADH (this is similar to your [ADH] exp)
38Procedure (Page 143)1 Step 1-6. Will create a kinetic curve for LDH (adjust volume of buffer to make 3.2ml)Similar to ADH2. Repeat kinetic curve with different concentrations of enzymeThis is protocol you will use as you purify LDHDo this assay on the unknown samples from step one and 2a from group 1.
39C2-3. Page 144Table C.2-3. Lactate Dehydrogenase Reaction Time CoursesReading numbertime(seconds)A340 readings50 mlsample100 ml sample200 ml sample300 ml sample400 ml sample121533044556067579081059120
40Next 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
41Next labNeed member of group to be here at 1:30 to begin washing columnWill need to measure absorbance at 280 to determine that contaminating protein is lost from column. Wash and measure until A280 is constant.
42StrategyFor samples generated determine amount of protein (A280 ) and activityActivity per microgram of protein =s specific activityYou strive for maximal activity per unit of protein. (table C2-4 Column G, Page 162)
44Will fill out this critical table as we proceed page 162 (day 4) Table C.2-4. Enzyme Purification TableNet volume(ml)V0 units per mlV0 unitsTotal(an “amount”)Protein content(% of total)Proteinconcentration(mg/ml)Net amountof protein(mg)Specific Activity(V0/mg protein)StepABCDEFG1. Cleared2. (NH4)2SO4 Supernatant3. diluted dialyzed sample/ solution placed on column4. pooled peak tubes from columnColumn 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
45This Lab 4 lab periods Prelab= 12 points Lab Report= 50 points First exam in period 4