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

Jun 8. 2016


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?


First, it is essential 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 a 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 optimized for MS protein ID [1,2] (see examples below).

Increase protein concentration on PVDF membrane

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?


[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
  • 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

About Alphalyse

Alphalyse uses expertise in the field of protein chemistry and bioinformatics, combined with top-of-the-line mass spectrometry equipment, to provide protein analysis services globally.

Alphalyse aims to deliver high quality data and customer service to all of our clients in a fast and convenient way.

Newsletter Subscribe to The Alphalyse Newsletter and get more
information on protein analysis methods and applications
© 2023 Alphalyse, Inc.