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Enzymatic Digestion of Proteins in Solution and in SDS Polyacrylamide Gels

  • Kathryn L. Stone
  • Kenneth R. Williams
Protocol
Part of the Springer Protocols Handbooks book series (SPH)

Abstract

Although most prokaryotic proteins have free NH2-termini and therefore can be directly sequenced, most eukaryotic proteins have blocked NH2-termini which precludes Edman degradation (1,2). In these instances, one of the most direct approaches to obtaining partial amino acid sequences is via enzymatic or chemical cleavage followed by peptide fractionation and sequencing. Although several different approaches may be taken to cleave proteins, one of the most common is to digest the protein enzymatically with a relatively specific protease such as trypsin or lysyl endopeptidase. Since final purification is often dependent on SDS-PAGE, cleavage procedures that can either be carried out in the polyacrylamide gel matrix (3,4) or that may be used on samples that have been blotted from SDS polyacrylamide gels onto PVDF (5) or nitrocellulose (5,6 membranes are extremely useful. In both instances, the proteins are usually stained with Coomassie blue or Ponceau S prior to excision, and proteolytic digestion and the resulting peptides are separated by reverse-phase HPLC. Relatively straightforward solution and in-gel digestion procedures that have been used extensively in the Keck Foundation Biotechnology Resource Laboratory at Yale University will be described in this chapter, whereas a procedure suitable for in situ digestion of SDS-PAGE blotted proteins is described in  Chapter 77.

Keywords

Amino Acid Analysis Iodoacetic Acid Partial Amino Acid Sequence Lysyl Endopeptidase Relative Staining Intensity 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

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Copyright information

© Humana Press Inc., Totowa, NJ 2002

Authors and Affiliations

  • Kathryn L. Stone
    • 1
  • Kenneth R. Williams
    • 1
  1. 1.Yale Cancer Center Mass Spectrometry Resource and W. M. Keck Foundation Biotechnology Resource LaboratoryNew Haven

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