Protein Protocols Protein Protocols

Enzymatic Digestion of Membrane-Bound Protein 523
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Enzymatic Digestion of Membrane-Bound Proteins
for Peptide Mapping and Internal Sequence Analysis
Joseph Fernandez and Sheenah M. Mische
1. Introduction
Enzymatic digestion of membrane-bound proteins is a sensitive procedure for
obtaining internal sequence data of proteins that either have a blocked amino terminus
or require two or more stretches of sequence data for DNA cloning or confirmation of
protein identification. Since the final step of protein purification is usually SDS-PAGE,
electroblotting to either PVDF or nitrocellulose is the simplest and most common procedure
for recovering protein free of contaminants (SDS, acrylamide, and so forth)
with a high yield. The first report for enzymatic digestion of a nitrocellulose-bound
protein for internal sequence analysis was by Aebersold et al. in 1987, with a more
detailed procedure later reported by Tempst et al. in 1990 (1,2). Basically, these procedures
first treated the nitrocellulose-bound protein with PVP-40 (polyvinyl
pyrrolidone, M r 40,000) to prevent enzyme adsorption to any remaining nonspecific
protein binding sites on the membrane, washed extensively to remove excess PVP-40,
and the sample was enzymatically digested at 37°C overnight. Attempts with PVDFbound
protein using the above procedures (3,4) give poor results and generally require
>25 µg of protein. PVDF is preferred over nitrocellulose because it can be used for a
variety of other structural analysis procedures, such as amino-terminal sequence analysis
and amino acid analysis. In addition, peptide recovery from PVDF-bound protein is
higher, particularly from higher retention PVDF (ProBlott, Westran, Immobilon P sq ).
Finally, PVDF-bound protein can be stored dry as opposed to nitrocellulose, which
must remain wet during storage and work up to prevent losses during digestion.
Enzymatic digestion of both PVDF- and nitrocellulose-bound protein in the presence
of 1% hydrogenated Triton X-100 (RTX-100) buffers as listed in Table 1 was
first performed after treating the protein band with PVP-40 (5). Unfortunately, the
RTX-100 buffer also removes PVP-40 from the membrane, which can interfere with
subsequent reverse-phase HPLC. Further studies (6,7) demonstrate that treatment with
PVP-40 is unnecessary when RTX-100 is used in the digestion buffer. It appears that
RTX-100 acts as both a blocking reagent and a strong elution reagent.
PVDF-bound proteins are visualized by staining and subsequently excised from the
blot. Protein bands are immersed in hydrogenated Triton X-100 (RTX-100), which
From: The Protein Protocols Handbook, 2nd Edition
Edited by: J. M. Walker © Humana Press Inc., Totowa, NJ
523