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Western Blot Forum Discuss western blotting and immunoblot in the western blot forum discussion board. Ask Questions about transfers, blocking, membrane antibody incubation and exposures.


Western Blot Problem

Western Blot Problem - Western Blot Forum

Western Blot Problem - Discuss western blotting and immunoblot in the western blot forum discussion board. Ask Questions about transfers, blocking, membrane antibody incubation and exposures.


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  #1  
Old 06-29-2008, 11:50 AM
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Default Western Blot Problem



I have a problem with western blot, if you can guide me to figure out what the problem is I will be very appreciated.

Normally when I try to run the samples it takes about 10 hours to be done, Is it a problem in the buffer?

the reciepe that I use for the preparation of the running buffer is :

15.1 g Tris base
94 g Glycine
50 ml 10% SDS
adjust the Ph(8.3)
complete the volume to one litre

I adjust the current on 50 mA then on 114 mA. we don't adust the voltage but it was low.
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Old 07-01-2008, 06:39 PM
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Default Re: Western Blot Problem

You do have a problem. I use semi-dry transfer and am done after 19 minutes, at 0.52A (constant), 17V.
When I used tank transfer I ran at 100V(constant), 40-60 minutes, no limit on amps, with a stirrer in a cold room (no limit on amps).

My buffer, also TGS is ready-made but here is the recipe:
1X TGS
25mM Tris
192mM Glycine
0.1% SDS
pH 8.3
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Old 07-28-2009, 06:55 AM
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Default Re: Western Blot Problem

My running buffer:
Gly 14.4 g
Tris 3.0 g
SDS 1 g
volume 1 L

use constant voltage 60 V, about 3 h.
Or the stacking gel --85 V, then the sep gel --110 V, about 2 h
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