1. Proteomics Module Day 1 Tech talk

2. Experiment: Yeast protein expression changes caused by H 2 O 2 exposure. ► 2 Control groups (A and B): nothing added ► 2 experiment groups (C and D): 1 hour incubation with 0.5 mM H 2 O 2 ► 1 experiment group (E): 2 hour incubation with 0.5 mM H 2 O 2 ► Grow yeast culture overnight: log phase growth ► Extract soluble proteins and use 2D gel electrophoresis and mass- spectrometry to identify proteins with altered expression

3. Lab Safety Issues ► Working with baker’s yeast (Sacchromyces cerevisiae): a non- pathogen ► Some chemicals are toxic: be careful ► Wear lab coat, gloves and eye protection ► Dispose of materials in appropriate receptacles ► Keep work area clean and neat ► Be aware of neighbors: don’t splash ► Share centrifuges and other lab instruments ► Follow all standard laboratory procedures for your course

4. Technical Tips Check off each step in protocols as you do them Label tubes carefully and completely—top and side  Name or group identifier  Sample identification  Date  Concentration if appropriate Keep tube lids closed  Dust and skin proteins will contaminate sample Pipetting issues  Make sure volume is set correctly  When measuring, depress plunger to first stop  When delivering, depress plunger to second stop

5. Day 1: Harvest Soluble Proteins from Yeast ► Collect yeast by centrifugation—discard media ► Extract soluble proteins using YeastBuster reagent ► Centrifuge to pellet membranes and non-dissolved debris ► Collect supernatant containing soluble proteins ► Save samples for protein assay and 1D gel electrophoresis For 2D gel sample ► Precipitate proteins ► Wash proteins ► Dry and freeze

6. Harvesting yeast proteins: getting soluble proteins out of the yeast ► Yeast have a thick cell wall as well as a cell membrane which makes it difficult to extract proteins ► YeastBuster reagent will do the trick  Lithium chloride and ethylene glycol to make membrane permeable  THP (tris(hydroxypropyl)phosphine) a reducing agent to de-stablize the cell wall  Protease inhibitors to inhibit yeast proteases and preserve the proteins we want  Nuclease to break down large DNA and RNA polymers and make solutions less viscous

7. Protein Sample Separation Protein Concentration Determination

8. Precipitation of Soluble Proteins ► Precipitation will separate proteins from other soluble cell materials such as nucleic acids, organic compounds, YeastBuster reagents, etc. ► Use an acid/ketone mixture (proprietary combination) to precipitate soluble proteins (trichloroacetic acid/acetone is often used) ► Wash the protein pellet to get rid of non-protein contaminants ► Dissolve proteins in a water based buffer

9. Protein Concentration: Bio-Rad RCDC Protein Assay ► Transfer 10.0 ul of sample from your PC1 and PC2 tube to a new tube ► Add 15.0 ul ddwater to each tube. ► Add 125 ul RC Reagent I –mix 1minute ► Add 125 ul RC Reagent II Mix. Centrifuge at high speed for 5 minutes. ► Remove supernatant ► Add 127 ul Working Reagent A and wait 5 minutes ► Add 1,000 ul DC Reagent B. Incubate for 15 minutes on bench ► Read the absorbance at 600 nm using Spectrophotometer ► Instructor use prepare a set of standards to generate a standard curve

10. Samples at the end of Day 1 ► 1D: 12 ul in.5 ml tube for 1D SDS-Page gel electrophoresis on day 2 ► 2D: washed and dried proteins in freezer. This sample will be used for 2D gel protocols and mass-spectrometry analysis.