IUBio

SDSPAGE Problems

Dr E. Buxbaum EB15 at le.ac.uk
Tue Jan 6 21:08:12 EST 1998


Fergus Doherty wrote:

> "Here in the lab I have an annoying problem with my gels. I'm using
> the protocols you gave me and they were OK always. I used those with
> success in Bath as well. But now, despite I use fresh precisely made
> solutions, there isn't a proper front line of my gel 

The following possible explanations come to mind:

1) High salt content of sample. Desalt, for example by
chloroform/methanol precipitation.

2) pH of gel buffers changed over time. Prepare fresh buffers.

3) Gels to old. A batch of gels can be stored in the fridge for about 2
weeks with few problems. After that, diffusion will eliminate the pH
jump between stacking and separating gel. Prepare fresh gels.

4) Comb slightly thicker than spacers or spacers of different thickness.
This results in a gap between gel and glas plate and this leads to the
observed effect. Use proper comb/spacer combinations. Some manufacturers
use different colours for different thickness, this prevents mixing.

5) Stacking gel buffer used for separating gel and vice versa. 

6) Decomposition of acrylamide to acrylic acid results in increased
conductivity. Acrylic acid can be removed by adding some ion exchanger
to the acrylamide stock solution. Prepare (or buy) acrylamide stock in
small batches and store in the fridge. Discard if too old.



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