Western Blotting Problem

Mehdi Alimadadi mehdia at interchange.ubc.ca
Fri Jan 7 03:47:17 EST 2000


Dear Netters

I hope you all be fine and happy Y2OK.
Please kindly advise me on the following problem if you can think of any
idea:

- When I am running the SDS-PAGE to get the gel for protein separation I can
reach the Voltage even more than 200 but when I want to transfer the protein
to the membrane I can hardly go for more than 10 Volts with the same power
supply, so naturally to have a good transfer I should leave it overnight
while it can be done in one hour with voltage of 100 V.  What could be the
reason?  Could it be the transfer buffer (I think I should dilute that
more)?  If so
please provide me with a suitable transfer buffer ingredients (in terms of
the chemicals and the amounts and the pH).

- What solution I should use to have the sensitivity of 100 ng/ml detection
(DAB & urea peroxidase or immidazol & nickel, or ...., please provide me
with the amounts as well or pH if needed)?

Thanks in advance.

Mehdi Alimadadi
E-mail: mehdia at interchange.ubc.ca














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