Protein Sequencing

N-terminal protein sequence analysis

N-terminal protein sequence analysis is performed by automated Edman degradation. Quantitation of all residues is possible at the 5-10 pmol level. Cys is determined as the Cys (carboxamidomethyl) derivatives formed following reduction and alkylation of the protein or peptide sample. Starting with as little as 10 pmol of material, 25 residues can routinely be unambiguously assigned. Many of our samples come to us as transblots of proteins onto polyvinylidene difluoride (PVDF) membranes. This powerful technique combines polyacrylamide gel electrophoresis and electrophoretic transfer, and allows us to sequence targeted proteins in a complex mixture. With as little as a 60 pmol transblotted sample, as many as 15-20 residues can usually be unambiguously assigned. New technologies also allow us to directly analyze individual protein bands excised from SDS-PAGE gels and passively eluted. The protein sample in this buffer is applied directly to the sequencer cartridge, eliminating the need for extensive purification or transblot of the protein to PVDF membrane. CBI can perform the passive elution or transblot procedures for an additional cost. Direct sequencing of synthetic peptides still bound to their synthesis resin is also possible.

C-terminal protein sequence analysis

C-terminal protein sequence analysis can be accomplished by carboxypeptidase Y digestion. The substrate protein is digested with carboxypeptidase Y and the amino acids that are released are quantitated by amino acid analysis, usually at five (5) time points during the digestion. A minimum of 2 nmol of protein is needed. Usually, one to four residues can be sequenced and identified.


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Commonwealth Biotechnologies, Inc. 601 Biotech Drive, Richmond, VA 23235 800-735-9224 (800-RELY-CBI) FAX: 804-648-2641 email: [email protected]