Proteomics Module Day 1 Tech talk. 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
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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
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
Check off each step in protocols as you do them
Label tubes carefully and completely—top and side
Keep tube lids closed
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
Dry and freeze
Protein Concentration Determination
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
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.
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
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.