How should I prepare my samples for 2D electrophoresis, and how much protein should I load to get protein ID by mass spec?
Protein samples for 2D PAGE should not contain high salt concentrations, ionic detergents like SDS, or cellular debris and DNA from cell lysis, as these will disturb isoelectric focussing (IEF) and cause spot streaking in the gel [1,2].
A general 2D gel sample preparation protocol could include the steps:
- Use the lysis procedure and buffer that you have used before
- Clean up and concentrate the sample, for example using the GE 2D clean-up kit.
- Dissolve the proteins in 2D lysis buffer (8.9M urea, 2% Triton X-100, 0.5% IPG buffer, 0.13M DTT and 8mM PMSF). This buffer is directly compatible with the 2D electrophoresis.
- Determine the protein concentration, for example using the 2D Quant kit.
The protein concentration should be approximately 1-20 ug/ul.
The protein load on each gel should be approximately 50-300 microgram for complex samples with many proteins, and 5-50 microgram for pure protein samples. For protein identification by mass spectrometry, the gels can be stained by standard Coomassie staining methods, or by more sensitive silver staining methods that have been optimized for MS protein ID [1,2].
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Alphalyse 2D gel services
Silver staining optimized for protein identification by mass spectrometry
 Wiśniewski et al: “Universal sample preparation method for proteome analysis“, Nature Methods, 2009
 Gundry et al: “Preparation of Proteins and Peptides for Mass Spectrometry Analysis in a Bottom-Up Proteomics Workflow“, Current Protocols in Molecular Biology, 2010