Problem with Polyacrylamide gel

[Dr Engelbert Buxbaum]

Am 14.05.2007, 19:44 Uhr, schrieb Bean Long  
<ben.long from yourfinger.anu.edu.au>:

> I agree Stew.  I have to admit I know many great scientists who don't  
> actually understand the basics of how certain pieces of equipment work  
> or how to do a particular experiment, but they ask the right questions  
> and provide the right interpretations.  These people are the ones  
> running large lab groups and looking to the frontiers of science.  I  
> truly understand your position too DK since, as much as I admire such  
> great scientists their lack of understanding of some basics really sh!ts 
> me to tears!!  As I see it, we have two choices as scientists. 1  
> Understand everything we do and get little done or, 2. Understand what  
> we need in order to get done what needs to be done!

But without a good understanding of a method you may not be able to  
interprete the results or see problems (leave alone solving them).

For example glycoproteins tend to run as broad smears in SDS-PAGE. The  
reason is that the sugar side chains are inhomogeneous and contain  
variable numbers of negative groups. One possible solution is to use a  
positive detergent like CTAB instead of the negatively charged SDS. Many  
people have tried that, but because they did not understand the  
electrochemistry behind DISC-electrophoresis they used buffers designed  
for anodic electrophoresis in a cathodic system. Result: no stacking and  
the bands are still broad. The problem can (and has been) solved, but that  
requires that the method is understood first.

So if you want to save time by using precast gels, by all means do so. But  
make sure you can cast your own first, and before even trying to do that  
make sure you understand how the method works.

To OP: thorough degassing (either under vacuum or in an ultrasonic water  
bath) and thorough mixing of the gel solution should help. Also make sure  
that the gel has no contact with air. In the case of the separating gel  
that's easy, overlay with water-saturated n-buthanol. In the case of the  
stacking gel, make sure that your combs are shaped correctly. Both the  
Hoefer (now part of GE) and BioRad combs are cut out to deep and allow air  
access to the gel. This results in ill-shaped sample wells. I had proper  
combs cut in our workshop to solve that problem.