[Protein-analysis] western blotting help

leelavati shetty via proteins%40net.bio.net (by shettylv At gmail.com)
Fri Jan 5 21:45:11 EST 2007


We use wet blot transfer for our proteins the molecular weight of which is
18 -19 KDa. We use PVDF membrane for transfer and tris glycine SDS buffer
contg methanol 20%. Our gel is 15% Stacking and 5% resolving gel 10 x 10.5cm
1.5 mm thick.

The bands on the membrane are faint and no bands remain on the gel. We also
get some additional high molecular weight bands on the gel which we suspect
to be dimmer, but sometimes it is not visible clearly and sometimes we get
good result. Is the transfer not efficient. We use alkaline phosphatase NBT
and BCIP for detection.

Can anyone *guide me on a good protocol* for western blotting wet transfer
method using NBT and BCIP. Also what condition shall I use for transfer
since we do overnight transfers, can I can reduce the time

Also can I store my antibody used once and reuse it second time.

-- 
Leelavati Shetty


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