Western Blotting problems (Alsagaby, Suliman)

[Nikola Wenta]

Hi!
As you are using a PVDF membran, did you wet the membrane with methanol?
You didn't stain the gel with Coomassie before blotting, right? Are you
sure that you really had protein on your gel (Ponceau)? Do you use
semi-dry blot? Than you need to take care that you have your gel on top
of the membrane.
Niko


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> Message: 1
> Date: Sat, 13 Jun 2009 19:16:37 +0100
> From: "Alsagaby, Suliman" <salsag from essex.ac.uk>
> Subject: Western Blotting problems
> To: <methods from magpie.bio.indiana.edu>
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> Hello
> 
> I did western blot twice and every time I have the same 
> problem in which the PVDF membrane does not contain any 
> protein although it contains protein marker. The bands were 
> visualized by staining the gel. So it seems to me that the 
> bands did not transfer to the membrane. I verified the 
> transfer duration: one for 12h at 30V and the other one was 
> for 1h at 100V.
> 
> I am great full if any body can help me and direct me to the 
> right protocol if I am doing wrong one. 
> 
> 
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