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

2D PAGE preparation for MALDI ISD
Alphalyse
Sep 18. 2016

Question:

Should I use 1D or 2D SDS PAGE?

I’m interested in isolating and identifying cell surface receptor proteins.

In my experience, they typically have high MW, are glycosylated, and have transmembrane domains.

I’m considering 2D electrophoresis, but do you have any recommendations?

Answer:

2D PAGE is the classical approach to separating and visualizing many proteins from complex proteomics samples. It can give you an idea of the sample purity, which is useful before running mass spectrometry analyses relying on a sample with relatively high chromatographic purity (>80%), such as intact mass determination or peptide mapping. However, 2D PAGE also has some known disadvantages.

For example, you will rarely observe hydrophobic membrane proteins because they precipitate during IEF. So because of this, you will most likely only observe proteins within the pI range of the gel, typically pH 4-7, or 3-10, and the MW range of proteins in the gel is approximately 10-130 kDa [1-3].

Therefore, for large and hydrophobic proteins, it is better to make use of 1D SDS PAGE. The main reason is that you can dissolve the proteins in the 1D SDS PAGE buffer containing 0.1% SDS. But in addition, the gels have no pI limits, and the MW range can go up as high as 1.000 kDa [3].

Another possibility is to use in-solution digestion of the protein mixture. Typically, you would then perform protein identification of hundreds or even thousands of proteins by LC-MS/MS and a subsequent database search in Swiss-Prot/UniProtKB [1-3]. This approach is the alternative to running a gel and cutting out the bands of interest, prior to in-gel digestion and protein ID of each band. 

Related blog posts:

If you are considering to send samples for mass spec services, I also recommend that you take some time to check out these other blog posts for more information on how to optimize the use of SDS gels:

What you can do to increase the protein concentration before loading

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

1d-sds-page-2d-page-electrophoresis

References:

[1]          Sickmann et al: “The proteome of Saccharomyces cerevisiae mitochondria“, Proceedings of the National Academy of Sciences of the United States of America (PNAS), 2003

[2]          Andersen et al: “Directed Proteomic Analysis of the Human Nucleolus“, Current Biology, 2002

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

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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