Blurry bands ... - (Jul/07/2012 )

mdfenko on Wed Jul 11 15:21:19 2012 said:

Thanks for the protocol .. I've already found a similar one online and already tried the run yesterday ...
but unfortunately, it totally failed ... I completely overlooked the fact that you run it at low voltage for quite a few hours. So after realizing that my running front was basically not moving, I increased the voltage ... which in fact was not a good idea ... so I ended up with a totally screwed gel .. not telling me anything ... but I'll repeat it with an overnight run at low voltage

... do I have to cool the buffer or can I run it at room temperature overnight?

BTW: How can I understand your gel preparation? What are the 10% spacer and the 10% running good for? And do I need them? And what would the size
be? And what do you mean with (runs “–“ to “+”)?


Thanks!

it is run at room temperature.

the original formulation had a 3 part gel: stack, spacer, and running gel. the 10% running gel is good for separating proteins in your size range. the 16.5% running gel is good for smaller proteins and peptides (we used it for neuropeptides including enkephalins).

we found that we could omit the spacer gel if we run a gradient gel.

if you look at the original paper, it gives the length that they worked out for each layer (you can scale them to your dimensions).

runs "-" to "+" is to tell the order of the electrodes (this is the standard configuration).

here is a later version of the protocol (with explanations):

That's really nice! Thanks a lot! I appreciate your help!