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Proteomics Module Day 1 Tech talk

Proteomics Module Day 1 Tech talk. 10 students in 5 groups of 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

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Proteomics Module Day 1 Tech talk

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  1. Proteomics ModuleDay 1 Tech talk 10 students in 5 groups of 2

  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 mMH2O2 • 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 when handling wet samples • 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

  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 activities 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. 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

  8. Samples at the end of Day 1 • 1D: 12 ul in .5 ml tube for 1D SDS-Page gel electrophoresis on day 2 • Q: about 38 ul in 1.7 ml tube for protein assays on Thursday • 2D: washed and dried proteins in freezer. This sample will be used for 2D gel protocols and mass-spectrometry analysis.

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