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

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

Question:

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

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 separate and visualize many proteins from complex proteomics samples. However, 2D PAGE has some known disadvantages. As an example, hydrophobic membrane proteins precipitate during IEF and are almost never observed. So most likely, you will 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].
 
For large and hydrophobic proteins it is therefore better to use 1D SDS PAGE. Mainly because the proteins can be dissolved in the 1D SDS PAGE buffer containing 0.1% SDS. 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. You would typically then perform protein identification by LC-MS/MS [1-3].
 
 

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

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.

www.alphalyse.com

Newsletter Subscribe to The Alphalyse Newsletter and get more
information on protein analysis methods and applications
© 2019 Alphalyse, Inc.