Proteomics ModuleProteomics ModuleDay 1 Tech talkDay 1 Tech talk

Experiment: Yeast protein expression Experiment: Yeast protein expression changes caused by Hchanges caused by H22OO2 2 exposure. exposure.

► 2 Control groups (A and B): nothing added2 Control groups (A and B): nothing added

► 2 experiment groups (C and D): 1 hour incubation with 0.5 mM 2 experiment groups (C and D): 1 hour incubation with 0.5 mM HH22OO22

► 1 experiment group (E): 2 hour incubation with 0.5 mM H1 experiment group (E): 2 hour incubation with 0.5 mM H22OO22

► Grow yeast culture overnight: log phase growthGrow yeast culture overnight: log phase growth

► Extract soluble proteins and use 2D gel electrophoresis and Extract soluble proteins and use 2D gel electrophoresis and mass-spectrometry to identify proteins with altered expressionmass-spectrometry to identify proteins with altered expression

Lab SafetyLab Safety IssuesIssues► Working with baker’s yeast (Working with baker’s yeast (Sacchromyces cerevisiaeSacchromyces cerevisiae): a non-): a non-

pathogenpathogen

► Some chemicals are toxic: be careful Some chemicals are toxic: be careful

► Wear lab coat, gloves and eye protectionWear lab coat, gloves and eye protection

► Dispose of materials in appropriate receptaclesDispose of materials in appropriate receptacles

► Keep work area clean and neatKeep work area clean and neat

► Be aware of neighbors: don’t splashBe aware of neighbors: don’t splash

► Share centrifuges and other lab instrumentsShare centrifuges and other lab instruments

► Follow all standard laboratory procedures for your courseFollow all standard laboratory procedures for your course

Technical TipsTechnical TipsCheck off each step in protocols as you do themCheck off each step in protocols as you do them

Label tubes carefully and completely—top and sideLabel tubes carefully and completely—top and side Name or group identifierName or group identifier Sample identificationSample identification DateDate Concentration if appropriateConcentration if appropriate

Keep tube lids closedKeep tube lids closed Dust and skin proteins will contaminate sampleDust and skin proteins will contaminate sample

Pipetting issuesPipetting issues Make sure volume is set correctlyMake sure volume is set correctly When measuring, depress plunger to first stopWhen measuring, depress plunger to first stop When delivering, depress plunger to second stopWhen delivering, depress plunger to second stop

Day 1: Day 1: Harvest Soluble Proteins from YeastHarvest Soluble Proteins from Yeast

► Collect yeast by centrifugation—discard mediaCollect yeast by centrifugation—discard media

► Extract soluble proteins using YeastBuster reagentExtract soluble proteins using YeastBuster reagent

► Centrifuge to pellet membranes and non-dissolved debrisCentrifuge to pellet membranes and non-dissolved debris

► Collect supernatant containing soluble proteinsCollect supernatant containing soluble proteins

► Save samples for protein assay and 1D gel electrophoresisSave samples for protein assay and 1D gel electrophoresisFor 2D gel sampleFor 2D gel sample

► Precipitate proteinsPrecipitate proteins

► Wash proteinsWash proteins

► Dry and freezeDry and freeze

Harvesting yeast proteins: getting Harvesting yeast proteins: getting

soluble proteins out of the yeastsoluble proteins out of the yeast

► Yeast have a thick cell wall as well as a cell membrane Yeast have a thick cell wall as well as a cell membrane which makes it difficult to extract proteinswhich makes it difficult to extract proteins

► YeastBuster reagent will do the trickYeastBuster reagent will do the trick

Lithium chloride and ethylene glycol to make membrane Lithium chloride and ethylene glycol to make membrane permeable permeable

THP (tris(hydroxypropyl)phosphine) a reducing agent to de-stablize THP (tris(hydroxypropyl)phosphine) a reducing agent to de-stablize the cell wall the cell wall

Protease inhibitors to inhibit yeast proteases and preserve the Protease inhibitors to inhibit yeast proteases and preserve the proteins we wantproteins we want

Nuclease to break down large DNA and RNA polymers and make Nuclease to break down large DNA and RNA polymers and make solutions less viscoussolutions less viscous

Protein Sample SeparationProtein Sample Separation

Protein Concentration Determination

Precipitation of Soluble ProteinsPrecipitation of Soluble Proteins

► Precipitation will separate proteins from other soluble cell Precipitation will separate proteins from other soluble cell materials such as nucleic acids, organic compounds, materials such as nucleic acids, organic compounds, YeastBuster reagents, etc.YeastBuster reagents, etc.

► Use an acid/ketone mixture (proprietary combination) to Use an acid/ketone mixture (proprietary combination) to precipitate soluble proteins (trichloroacetic acid/acetone is precipitate soluble proteins (trichloroacetic acid/acetone is often used)often used)

► Wash the protein pellet to get rid of non-protein Wash the protein pellet to get rid of non-protein contaminantscontaminants

► Dissolve proteins in a water based bufferDissolve proteins in a water based buffer

Protein Concentration:Protein Concentration: Bio-Rad RCDC Protein Bio-Rad RCDC Protein

AssayAssay ► Transfer 10.0 ul of sample from your PC1 and PC2 tube to a new Transfer 10.0 ul of sample from your PC1 and PC2 tube to a new

tubetube

► Add 15.0 ul ddwater to each tube. Add 15.0 ul ddwater to each tube.

► Add 125 ul RC Reagent I –mix 1minuteAdd 125 ul RC Reagent I –mix 1minute

► Add 125 ul RC Reagent II Mix. Centrifuge at high speed for 5 Add 125 ul RC Reagent II Mix. Centrifuge at high speed for 5 minutes.minutes.

► Remove supernatantRemove supernatant

► Add 127 ul Working Reagent A and wait 5 minutesAdd 127 ul Working Reagent A and wait 5 minutes

► Add 1,000 ul DC Reagent B. Incubate for 15 minutes on benchAdd 1,000 ul DC Reagent B. Incubate for 15 minutes on bench

► Read the absorbance at 600 nm using SpectrophotometerRead the absorbance at 600 nm using Spectrophotometer

► Instructor use prepare a set of standards to generate a standard Instructor use prepare a set of standards to generate a standard curvecurve

Samples at the end of Day 1Samples at the end of Day 1

► 1D: 12 ul in .5 ml tube for 1D SDS-Page gel 1D: 12 ul in .5 ml tube for 1D SDS-Page gel electrophoresis on day 2electrophoresis on day 2

► 2D: washed and dried proteins in freezer. This 2D: washed and dried proteins in freezer. This sample will be used for 2D gel protocols and sample will be used for 2D gel protocols and mass-spectrometry analysis.mass-spectrometry analysis.