western blotting help (leelavati shetty)

Hi,
1. What is your sample: cell extract, tissue extract?
2. What is the pore size of your PVDF? 0.45 or 0.22

I do westerns on 18KDa low abundance proteins from tissue extracts.
I use a 0.22ìm PVDF just to make sure that the proteins don't go "through" the membrane.
If you have the apparatus, try to use semi-dry transfer. People say that it works very well with small MW proteins and the big advantage is that it only takes 20min.

Sotiris Tsalavos



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Today's Topics:

1. western blotting help (leelavati shetty)


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Message: 1
Date: Sat, 6 Jan 2007 08:15:11 +0530
From: "leelavati shetty" <[Only registered users see links. ]>
Subject: [Protein-analysis] western blotting help
To: [Only registered users see links. ]
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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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