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small proteins have run ahead of tracking dye


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#1 polaris

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Posted 12 May 2010 - 09:24 AM

Hi all.

I run an acrylamide gel electrophoresis for RIPA and have encountered a new problem after preparing some new reagents. The smaller mol. weight proteins have run off the gel as if I let it run long, but the tracking dye had only just reached the bottom of the gel. Tracking dye was one of the new reagents. Any other likely candidates? I run at constant power (amps) and the voltage started and finished a little lower than usual - usually starts at ~107v and runs up to ~390V after 4 hours, but this time 97v to 335v after 4.5 hrs. I also decided to make my stacking gel a little taller - usually 1cm from well bottom to top of resolving gel, this time 2cm, but I've run it this way before without the run-off problem.

Thanks for any advice...

-P

#2 mdfenko

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Posted 18 May 2010 - 11:08 AM

what else is new?

check the pH of all of your buffers.

lower voltage indicates that the ionic strength is higher. the buffers may be made wrong (too strong).

these factors may affect mobility.
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