why gel is not running in straight line?? (View forum version)



porfirion

Posted 04 November 2009 - 08:29 AM

Hello,
I have a problem with electrophoresis, my gels (regular cell lysates) are not running in a straight line, but thay are one side curved-up . does anyone have some idea why it is happening??

Prep!

Posted 04 November 2009 - 08:39 AM

smily effect...
heat is not uniform across the gel...
try running at a lower voltage!!!
wat conditions are u running in???
is it just the dye showing this effect or the bands too???

mdfenko

Posted 04 November 2009 - 01:32 PM

dirty electrode.

phage434

Posted 04 November 2009 - 02:32 PM

Tilted gel tray with different buffer depths at each side.

swanny

Posted 04 November 2009 - 04:56 PM

Tilted gel tray with different buffer depths at each side.

or tilted gel tray when the gel is setting, which will lead to a gel of variable thickness.

Do you have a picture?

jiajia1987

Posted 04 November 2009 - 05:15 PM

It usually works ok for me when I decrease to a lower voltage.

miBunny

Posted 04 November 2009 - 06:56 PM

How much protien lysate per well?

Adrian K

Posted 04 November 2009 - 07:18 PM

this used to happen to me on the first and last well, I also cant figure it out why... maybe too high voltage is the culprit. My solution: i ignore the 1st and last well when i run my page gel...

porfirion

Posted 05 November 2009 - 12:03 AM

Thanx everyone for your posts.
I usually run it at 70V at the beggining and 120-130 later, is it to high?? samples contain between 20-40 ug of protein in reduced condition.

Prep!

Posted 05 November 2009 - 12:54 AM

Thanx everyone for your posts.
I usually run it at 70V at the beggining and 120-130 later, is it to high?? samples contain between 20-40 ug of protein in reduced condition.



why dont u try running it at a constant voltage... instead of switching.. might help..
and 40 mcg is too much for a SDS-PAGE.. tat too iof u are silver staining it!!!

mdfenko

Posted 05 November 2009 - 09:06 AM

this used to happen to me on the first and last well, I also cant figure it out why... maybe too high voltage is the culprit. My solution: i ignore the 1st and last well when i run my page gel...


this happens because the edges run cooler than the central portion of the gel.

lab rat

Posted 05 November 2009 - 12:34 PM

The temp of the gel can make a difference. Do you circulate your running buffer? If the tank is small, you can stick it in an ice bucket and it will be fine. If it's large, you can set it on a magnetic stirrer with a stir bar underneath and run it at a lower voltage, or use an attachment for a circulating cold water bath.

Adrian K

Posted 05 November 2009 - 07:56 PM

this used to happen to me on the first and last well, I also cant figure it out why... maybe too high voltage is the culprit. My solution: i ignore the 1st and last well when i run my page gel...


this happens because the edges run cooler than the central portion of the gel.



Oh....I didn't know that....haha... thank you for the enlightenment.
:rolleyes:

jiajia1987

Posted 09 November 2009 - 12:06 AM

Plus, avoid loading too much sample into one gel. You can always divide your sample volume into half and load them into two wells.