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Western Blotting Problem

Bernard Murray, PhD spam at 127.0.0.1
Fri Jan 7 23:03:49 EST 2000


In article <8549ar$6lt$1 at nntp.itservices.ubc.ca>, "Mehdi Alimadadi"
<mehdia at interchange.ubc.ca> wrote:

> - 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,

Not all high-voltage power packs are "high current".  Running an
electrophoresis minigel at 200 V only uses about 10 W (current of 50 mA)
but the electroblotting stage generates a much higher current.
Transferring a couple of minigels at 100 V gives a final current
of about 300 mA (so 30 W).  These numbers will vary with your own
equipment.  Many small power packs are limited to currents
of 200 mA or even 100 mA and so will run under current limitation
if you set them to a high voltage.

> please provide me with a suitable transfer buffer ingredients (in terms of
> the chemicals and the amounts and the pH).

Standard "Towbin" buffer (Tris/Glycine with 20% methanol) is just fine
for most purposes.
 
> - 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)?

The detection limit you indicate is meaningless.  Do you mean
100 ng per band?  I assume you are refering to detection after
immunoblotting (or otherwise you can just silver stain your gel).

HRP: Chloronapthol or chemiluminescent
AP: BCIP/NBT or chemiluminescent

DAB or benzidine are rarely used for blotting detection these
days.  Metal/DAB is more for immunohistochemistry.

   Bernard

-- 
Bernard P. Murray, PhD
bpmurray at cgl . ucsf . edu
Department of Cellular & Molecular Pharmacology, UCSF




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