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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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experiment yeast protein expression changes caused by h 2 o 2 exposure
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

lab safety issues
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

technical tips
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
day 1 harvest soluble proteins from yeast
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

harvesting yeast proteins getting soluble proteins out of the yeast
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
protein sample separation
Protein Sample Separation

Protein Concentration Determination

precipitation of soluble proteins
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

protein concentration bio rad rcdc protein assay
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

samples at the end of day 1
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.