Protocol for gel silver staining – mass spec & protein identification

Aug 6. 2015

Problems arising from using commercial staining kits:

Scientists use silver staining for sensitive detection of proteins separated by 1D and 2D SDS PAGE. The detection limits should be 0.5-5 ng [1, 2].

Many silver staining protocols and commercial staining kits are not compatible with mass spectrometry. This is because they use cross-linking reagents. Therefore, it is important with a silver stain protocol compatible with mass spec if one wishes to identify proteins (protein identification) from bands and spots isolated from SDS PAGE gels [1, 2].

Our recommendations:

For low protein amounts, we recommend that you use a silver staining method optimized for compatibility and sensitivity with trypsin digestion and MS analysis. Many silver staining protocols are not compatible and thus do not work with protein identification using mass spec (mass spectroscopy) [1, 2].

In the protocol (follow the link below), we describe our Alphalyse SDS Page gel silver staining protocol which we optimized for mass spectrometric analysis.

Get a confident protein identifications and high sequence coverage:

The protocol results in confident protein identifications. In addition, it provides a high sequence coverage by MALDI MS and ESI MS. This is due to a high recovery of peptides from the stained gel. The protocol has been tested and documented in many publications [3].

Get the full protocol here


[1]          Chevallet et al: Silver staining of proteins in polyacrylamide gels“, Nature Protocols2006

[2]          Rabilloud, T.: “Mechanisms of protein silver staining in polyacrylamide gels: a 10-year synthesis“, Electrophoresis1990

[3]          Mortz et al: Improved silver staining protocols for high sensitivity protein identification using matrix-assisted laser desorption/ionization-time of flight analysis“, Proteomics2001

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

  • Increase protein concentration on PVDF membrane

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

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

  • 2D PAGE preparation for MALDI ISD

    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
© 2021 Alphalyse, Inc.