SDS-page and the bottom of the gel - SDS-page (Nov/04/2007 )
Hi
I was wondering why the protiens does not float out into the running-buffer when they reach the bottom of an SDS-page?
I have been sorting histones and been careful about not letting the SDS-page run for longer time than necessary, but then I was told that the protiens does not float out into the runnig buffer, why not? I thought that the gel just endes like openings and the voltage would make the proteins keep moving toward the catode.
I hope you can help me.
Thanks
Bio_2007
If you run the gel long enough, the protein will run out of the gel.
I accidentally tried it once (forgotten about the gel)... and lost all my proteins and have a clear western blot ... I nearly cried...
what it means may be ... if the loading dye run off the gel, the proteins are still on the gel.
As the proteins are bigger and run much slower than the loading buffer.
Hope this may help.
I agree with Minnie, if you let it run too long, the proteins will go out of the gel, the smallest first, and then the biggest.
there are preparative gel electrophoresis apparati available (or used to be) that you run the sample all the way through the gel and elute from the bottom (or center if run radially as with the "bullseye" apparatus), washing the protein to fractions with a flow of (lower) electrode buffer.
we once took much hope in this method using "Prepcell"; in principle it worked but elutions were too diluted, and contained various (SDS-denaturized) proteins;
we used a preparative page apparatus made by tara (not sure if the company still exists, the guy who handmade them passed away).
we adjusted the elution flow to the slowest that allowed for separation of near proteins and maximized our protein load.
most of the time we used it for native page so there was no sds denaturing.
we adjusted the elution flow to the slowest that allowed for separation of near proteins and maximized our protein load.
most of the time we used it for native page so there was no sds denaturing.
if I think of using a preparative PAGE apparatus under native conditions, I wonder if I will need too much electric energy to get the system run but which may denaturized or lyse my proteins...
the apparatus is jacketed so we are able to cool the gel. but, you don't need to run it so fast that it would get excessively hot (we run it just like any other gel, with n mA/unit area). always check the manual that comes with the apparatus.
by the way, i could see concern for denaturing but i've never seen a protein lyse because of the electric field.