C.B.S. Scientific HTLE-7002 - Manual (Page 5)

C. B. S. œ Scientific 5 HTLE-7002
SECTION 1
General Information
1.1
Introduction
Peptide mapping is a powerful technique used in the fields of molecular biology and biochemistry
to help determine peptide structure and composition of proteins. Peptide maps or fingerprints of
proteolysed proteins are usually obtained by resolution on either one-dimensional SDS-PAGE
analysis (Cleveland), reverse-phase HPLC, or two-dimensional separation on thin layer plates.
Perhaps the most common applications of peptide mapping are: I) to reveal identities between
proteins suspected to be encoded by the same or related genes, ii) to prepare individual peptides
to determine amino acid composition and sequence, and iii) to determine the precise location of
amino acid residues that are post-translationally modified by either fatty acid acylation,
glycosylation, methylation, acetylation, or phosphorylation. Because the biochemist is often
faced with the reality of obtaining only vanishingly small amounts of a protein for analysis it is
often difficult or impossible to perform crucial experiments which reveal some of these important
characteristics.
Two-dimensional separation of proteolytic digests by electrophoresis and chromatography on thin
layer cellulose plates is a technique that is well suited to solve at least some of these problems.
First, it is an extremely sensitive technique that requires only small amounts metabolically labeled
product (only a few dpm). Second, because digests are resolved in two dimensions, a variety of
information is derived that often yields subtle but important clues about a given peptide that may
help reveal its composition. Finally, because cellulose is an inert substance, the peptide material
can be recovered for secondary analysis such as determining amino acid composition and
sequence or determining the presence and position of phosphoamino acid residues.
Peptide mapping experiments are often frustrating because of problems that can arise during
samples preparation leading to poor resolution and inconclusive results. However, for those
deciding to perform two-dimensional peptide maps on thin layer cellulose, the following is the
method used in our laboratory with repeated success. This technique focuses on the use of the
Hunter Thin Layer Electrophoresis system (HTLE-7002) to resolve peptides in the first dimension.
It features a unique clamping system and an inflatable nylon air bag to remove excess liquid from
the surface of the plate and prevent buffer from pooling on the plate. This creates and maintains
an ultrathin interface between the buffer and cellulose during high-voltage electrophoresis and
allows uniform cooling because of close and even contact with the cooling surface. Since it relies
on careful preparation of the sample, we have added detailed procedures on sample preparation
including illustrations and diagrams.
The protocols and discussions outlined below are the cumulative effort of many workers at the
Salk Institute who have refined the method originally employed by Wade Gibson (4). These
include: Jon Cooper, Suzanne Simon, Jill Meisenhelder, Kathy Gould, Clare Isacke, Jim
Woodgett, Ellen Freed, Gerry Weinmaster, David Meek, David Middlemas, Peter van der Geer
and Martin Broome.
Bill Boyle & Tony Hunter
Molecular Biology & Virology Laboratory
The Salk Institute For Biological Studies
La Jolla, California 92037