How much protein should I load for protein identification by mass spec

Alphalyse
Jun 8. 2016

Question:

How should I prepare my samples for 2D electrophoresis?

How should I prepare my samples for 2D electrophoresis, and how much protein should I load to get protein ID by the mass spec?

Answer:

First of all, it is important to know that protein samples for 2D PAGE should not contain high salt concentrations, ionic detergents like SDS, or cellular debris and DNA from cell lysis. These will disturb isoelectric focusing (IEF) and cause spot streaking in the gel [1,2], so stay clear of them.

As a rule of thumb, a general 2D gel sample preparation protocol should include the following steps:

  1. Continue to use the lysis procedure and buffer that you have used before.
  2. Clean up and concentrate the sample, for example, using the GE 2D clean-up kit.
  3. Then dissolve the proteins in 2D lysis buffer (8.9M urea, 2% Triton X-100, 0.5% IPG buffer, 0.13M DTT, and 8mM PMSF). I recommend using this buffer since it is directly compatible with the 2D electrophoresis.
  4. Finally, it would be best if you determined the protein concentration, for example, using the 2D Quant kit.

Ensure that the protein concentration is approximately 1-20 ug/ul.

I also recommend that the protein load on each gel should be approximately 50-300 micrograms for complex samples with many proteins and 5-50 micrograms for pure protein samples. Furthermore, 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] (see examples below).

Increase protein concentration on PVDF membrane

Find more information about 2d gel electrophoresis service:

Alphalyse performs 2D gel services and can advise you on how to prepare your samples. You can learn more on the website:

If you need silver staining optimized for protein identification by mass spectrometry, use this protocol, which was optimized for that particular purpose:

Recommended blog posts:

I have a couple of other blog posts which may be of inspiration for you when working with gel electrophoresis:

1D SDS PAGE versus 2D PAGE. Are there any recommendations on when to use which?

How much protein should I load for the best protein identification by mass spec?

References:

[1]          Wiśniewski et al.: “Universal sample preparation method for proteome analysis, “Nature Methods, 2009

[2]          Gundry et al.: “Preparation of Proteins and Peptides for Mass Spectrometry Analysis in a Bottom-Up Proteomics Workflow, “Current Protocols in Molecular Biology, 2010

2D PAGE preparation for MALDI ISD
More posts about 1D & 2D Electrophoresis
  • Protocol for gel silver staining – mass spec & protein identification

  • How to increase protein concentration before loading onto 1D SDS PAGE

  • How much protein should I load for protein identification by mass spec

  • 1D SDS PAGE versus 2D PAGE. Any recommendations?

  • Speedy and detailed protein ID of SDS gel bands

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