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In Gel Trypsin Digestion Protocol

What exactly is in gel trypsin digestion? Learn about In Gel Trypsin Digestion and the procedure for In Gel Trypsin Digestion.

Trypsin Digestion of Gel Slices: An Introduction

Polyacrylamide gel electrophoresis is a common lab procedure used to separate proteins in complex biological samples. The development of two-dimensional (2D) gel electrophoresis has provided a method to differentialy display proteins allowing the quantitative analysis of many hundreds to thousands of proteins at one time. By analyzing Differences in the 2D gel patterns between control and experimental conditions, one can now determine the effects on the expression of proteins and the synthesis of novel proteins under certain conditions such as cancer.

Tryptic Digestion of Peptides Directly in Polyacrylamide Gel Slices Protocol 

We explain in the following article how to directly digest proteins which have been run on a poly-acrylamide gel, and have been stained with Coomasie. By digesting the protein inside of the gel, you eliminate the need to elute the protein from the gel band slices or to transfer the protein of interest to a PVDF / nitrocellulose membrane.

This protocol does not use any detergents which allows a direct analysis of the in gel protein digestion by Liquid Chromatography Mass Spectrometry (LC-MS) without HPLC fractionation.

in gel trypsin digestion

 The detection limit of proteins which are digestion in-gel is about 1 pmol of protein. As coomasie staining has a similar detection limit, it is known that a faintly stained coomasie stained band is enough to detect by mass spectrometry.

  Shevchenko et al. published the digestion of silver stained gel analysis by nanospray mass spectrometry, and this protocol is based on that protocol.

 

In Gel Digestion References:

A. Shevchenko, M. Wilm, O. Vorm, and M. Mann. Mass spectrometric sequencing of proteins from silver-stained polyacrylamide gels. Analytical Chemistry 68:850-858 (1996).

 

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