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 The CNRU Mass Spectrometry Core offers Proteomics and Metabolomics workshops.
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SAMPLE STRATEGY FOR PROTEIN ANALYSIS USING 2DGEDetermine what lysis and rehydration buffers to use Specific protein Specific organelle, membrane, or other subset of proteins Protein Profile- as many proteins as possible ImmunoprecitationRun 1D gel and/or western blot to optimize the sample prep conditions and/or to verify the sample contains the proteins you anticipated Run pH 3-10 or 4-7 strip and small-format (11cm) gel to make certain lysis conditions are appropriate. Analytical protein load (~ 200 µg). Coomassie stain. Troubleshoot and re-evaluate experiments What exactly are you looking for? May want to run a variety of narrow range (pH 5-8 or 7-11) using small format May want to do mini-analysis using different parameters (eg untreated vs treated cells) to determine if at least some differential regulation is occurring Western blot may be helpful if looking for a particular protein May try peptide mapping using preparative load (~1mg) Scale up- Run SINGLE pH 3-10 or 4-7 strip and large gel to make certain conditions are appropriate ($$) What are the upper and lower loading limits? What stain will you be using? Run series of large format gels to determine if narrow or micro range of strips is appropriate and what pH range you want If looking for a large number of proteins: Run overlapping pH ranges: pH 3-6, pH 5-8, pH 7-10 Micro-range strips available: pH 4.5-5.5, etc Various extraction methods such as step-wise extraction can give multiple fractions in single protocol If looking for particular protein(s) Estimate pI and use narrow- or micro-range strip Use western blotting to verify presence of protein in lysis buffer and/or following 2nd dimension |
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