Proteomics Module Day 1 Tech talk

1. Proteomics ModuleDay 1 Tech talk

2. Experiment: Yeast protein expression changes caused by H2O2 exposure. 2 Control groups (A and B): nothing added 2 experiment groups (C and D): 1 hour incubation with 0.5 mM H2O2 1 experiment group (E): 2 hour incubation with 0.5 mM H2O2 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 SafetyIssues 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.