Reusing PVDF Electroblotted Protein Samples After N-Terminal Sequencing To Obtain Unique Internal Amino Acid Sequence

Abstract

This chapter discusses a simple, high sensitivity method to obtain N-terminal and internal protein sequence from the same sample. The cyanogen bromide (CNBr)/ ortho-phthalaldehyde (OPA) method to reuse PVDF samples involves three steps: 1) determination of the N-terminal sequence (if not blocked), 2) location of proline residues within CNBr fragments, and 3) isolation of unique sequence by delivering OPA when a proline becomes N-terminal during sequencing. CNBr in combination with OPA is a high sensitivity method to obtain N-terminal and limited internal sequence data. Methods that purify fragments for internal sequencing would have eliminated the N-terminal sequence determination necessary to verify a full-length clone and reduced the quantity available for sequencing even more. Reusing PVDF electroblotted protein samples allow obtaining N-terminal (when not blocked) and internal protein sequence data, consuming only protein that has been dedicated to N-terminal sequence determination.