western blot- biotin detection
charles_david at hms.harvard.edu
Fri Nov 15 15:07:13 EST 2002
i just tried posting this, then got an email saying it had failed. i'll try again.
sorry if this ends up being a double post.
We have been doing western blots to detect biotin-containing proteins
in our lab for a while. we use the biorad avidin-HRP conjugate. we
are usually looking at retinal pigment epithelium membranes, and have
been seeing what we assume to be endogenously biotinylated proteins
running at ~75 kD and ~120 kD. A search of the archives of this ng
reveals people have had a similar experience regarding endogenous
bands. anyway, the protocol biorad provides suggests we use TBS
buffer with 500 mM NaCl for all our washes/incubations. Recently one
of my coworkers has been using 150 mM NaCl, and an additional band
appears in his blots at around 55-60 kD. incidentally, the coomassie
stained gel also shows a major band at this region. my question is...
is this band likely to represent a biotinylated protein? is it
possible that the 500 mM wash is too stringent and we are ignoring a
biotinylated protein (following the bio-rad protocol)? If it is not
biotinylated (we are hoping it is not!) what kind of interaction might
it be? i really hope the 500 mM NaCl TBS washes is not causing us to
pass over some biotinylated proteins.
any help would be appreciated!
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