western blot help!!!
Dr Engelbert Buxbaum
engelbert_buxbaum at hotmail.com
Tue Apr 22 04:55:25 EST 2003
karthij96 at yahoo.com wrote:
> The transfer looks good--as i am able to see all the lanes & even the
> marker . The problem is
> that after i block the membrane in 5% milk ( carnation non-fat dry milk
> powder & made in 1X
> TBS) for an hour or even overnite in a cold room---the membrane refuses
> to wet---it looks as if
> its dry, only the egdes seem to be wet.
I am not sure what your problem is, as the membrane should not be
allowed to dry out during blocking. You should submerse your blott in
the blocking solution, and keep on a shaker or roller. After blocking
rinse for 2 times 2 min in TBS, then incubate with your antibody.
By the way, o/n blocking is not required, it only results in casein
layers forming over your protein bands and blocking access of your
antibodies to them, reducing sensitivity of your assay. In my experience
the best results (at least with Immobilon membrane) are obtained with
blocking times of 10-15 min, which is enough to prevent unspecific
binding of antibodies.
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