Horrendous background problem on chemiluminescent westerns
ckafer at iastate.edu
Tue Sep 8 14:29:22 EST 1998
Hi. I am hoping somebody can give some tips on cutting down the
background on my chemiluminescent westerns (ECL reagent). I get large
spots, patches and smears all over (as opposed to a nonspecific
recognition/banding pattern). Its particularly bad where the edges
of the gel contact the membrane during transfer.
I have tried just about everything I can think of. I wash extensively
(sometimes overnite!) with TBS-T after the secondary incubation.
Washes after the primary are for at least an hour with 4 changes of
large quantities of TBS-T. Both Ab incubations are done at rm temp.
in fresh blocking sol'n.
I've tried blocking w/ 3% BSA, 5% NF dry milk, 1-3% gelatin,
combinations of the above, and combinations of the above w/ various
concentrations of PVP. I have tried blocking at room temp, 37 deg, 4
deg overnite and combinations of these for 30 minutes to hours to
We use Biorad pure Nitrocellulose and I've tried MSI nitrocellulose.
The transfer is done in a tank w/ a standard buffer (Tris, glycine,
SDS, methanol) for an hour to overnite at low voltage.
Anybody have some magical blocking solution?? ANY other insights
would be appreciated too!
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