Internal Amino Acid Sequencing of Proteins Recovered from One- or Two-Dimensional Gels

Abstract

This chapter focuses on the internal amino acid sequencing of proteins recovered from one- or two-dimensional gels. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis, combined with electroblotting and automated Edman degradation is routinely used for protein purification and N-terminal amino acid sequence analysis. In addition, development of the amino acid sequencing technology toward higher sensitivities unexpectedly leads to cases where artifactual N-terminal blocking is noted. Internal amino acid sequences are obtained by either in-gel or on-membrane cleavage, followed by separation and sequencing of the peptides generated. These methods can be carried out successfully only when the protein concentration in the gel or on the membrane is sufficiently high. For low-abundance proteins, it is therefore necessary to use methods that allow concentration of proteins into small volumes or onto small membrane surfaces.