Western Blot Problem

[Denise]

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.

[danfive]

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

[97b1]

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