Kl. Hsi et al., VISUALIZATION OF PROTEINS BY MODIFICATION OF LYSINES, CYSTEINES, AND PHOSPHORYLATED SERINES FACILITATES SAMPLE PREPARATION FOR MICROSEQUENCING, Analytical biochemistry, 258(1), 1998, pp. 38-47
Citations number
13
Categorie Soggetti
Biology,"Biochemical Research Methods","Chemistry Analytical
A procedure for visualization and sensitive detection of protein durin
g sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)
and subsequent sample preparation for sequence analysis is described.
This procedure utilizes either fluorescent, or visible tags for certa
in amino acids in protein molecules, e.g., lysines modified with dansy
l/dabsyl chloride and cystines/cysteines or phosphorylated serines mod
ified with iodoacetamidofluorescein (I-15) after proper sample pretrea
tments. Modifications are performed prior to SDS-PAGE, eliminating the
need for fixing, staining, and destaining as required for the convent
ional procedures. After electrophoresis, the fluorescent or visible ba
nds are excised from the gel, homogenized in microcentrifuge tubes, an
d soaked in an appropriate buffer to release the separated proteins in
to solution, Enzymatic digestion can then be carried out in solution f
or better efficiency of digestion and recovery. The subsequent HPLC ma
pping and collection of protein digests are performed on PE Applied Bi
osystems Model 173A MicroBlotter. The separated peptides containing ta
gged amino acids are visible on the PVDF membrane and can be excised f
or direct sequence analysis. This approach has been employed for selec
tively isolating the lysine, cysteine, or phosphorylated serine contai
ning peptides using model proteins. The sequencing results of the pept
ides generated from premodified proteins demonstrate that this approac
h facilitates sample preparation for microsequence analysis at low pic
omole level, Overall. recoveries of 20-30% by sequencing initial yield
s have been achieved using our model proteins. (C) 1998 Academic Press
.