Proteomics Module
Day 1 Tech talk
Experiment: Yeast protein expression changes
caused by H2O2 exposure.
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2 Control groups (A and B): nothing added
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2 experiment groups (C and D): 1 hour incubation with 0.5 mM H2O2
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1 experiment group (E): 2 hour incubation with 0.5 mM H2O2
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Grow yeast culture overnight: log phase growth
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Extract soluble proteins and use 2D gel electrophoresis and massspectrometry to identify proteins with altered expression
Lab Safety Issues
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Working with baker’s yeast (Sacchromyces cerevisiae): a nonpathogen
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Some chemicals are toxic: be careful
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Wear lab coat, gloves and eye protection
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Dispose of materials in appropriate receptacles
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Keep work area clean and neat
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Be aware of neighbors: don’t splash
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Share centrifuges and other lab instruments
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Follow all standard laboratory procedures for your course
Technical Tips
Check off each step in protocols as you do them
Label tubes carefully and completely—top and side
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Name or group identifier
Sample identification
Date
Concentration if appropriate
Keep tube lids closed
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Dust and skin proteins will contaminate sample
Pipetting issues
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Make sure volume is set correctly
When measuring, depress plunger to first stop
When delivering, depress plunger to second stop
Day 1:
Harvest Soluble Proteins from Yeast
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Collect yeast by centrifugation—discard media
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Extract soluble proteins using YeastBuster reagent
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Centrifuge to pellet membranes and non-dissolved debris
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Collect supernatant containing soluble proteins
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Save samples for protein assay and 1D gel electrophoresis
For 2D gel sample
Precipitate proteins
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Wash proteins
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Dry and freeze
Harvesting yeast proteins: getting soluble
proteins out of the yeast
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Yeast have a thick cell wall as well as a cell membrane which
makes it difficult to extract proteins
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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
Protein Sample Separation
Protein Concentration
Determination
Precipitation of Soluble Proteins
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Precipitation will separate proteins from other soluble cell materials
such as nucleic acids, organic compounds, YeastBuster reagents, etc.
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Use an acid/ketone mixture (proprietary combination) to precipitate
soluble proteins (trichloroacetic acid/acetone is often used)
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Wash the protein pellet to get rid of non-protein contaminants
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Dissolve proteins in a water based buffer
Protein Concentration:
Bio-Rad RCDC Protein Assay
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Transfer 10.0 ul of sample from your PC1 and PC2 tube to a new tube
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Add 15.0 ul ddwater to each tube.
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Add 125 ul RC Reagent I –mix 1minute
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Add 125 ul RC Reagent II Mix. Centrifuge at high speed for 5 minutes.
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Remove supernatant
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Add 127 ul Working Reagent A and wait 5 minutes
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Add 1,000 ul DC Reagent B. Incubate for 15 minutes on bench
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Read the absorbance at 600 nm using Spectrophotometer
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Instructor use prepare a set of standards to generate a standard curve
Samples at the end of Day 1
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1D: 12 ul in .5 ml tube for 1D SDS-Page gel
electrophoresis on day 2
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2D: washed and dried proteins in freezer. This sample
will be used for 2D gel protocols and mass-spectrometry
analysis.