What's the problem with this SDS-PAGE?
Posted 28 July 2009 - 11:34 PM
Posted 29 July 2009 - 07:48 AM
Assuming there is nothing wrong with the gel itself, I think you ran too much lysate. Occasionally I get similar looking gels when I overload them. Typically, when I run crude lysates I run a lot less. I don't quantitate either, but my method is as follows: pellet 1 ml of bacterial culture, resuspend pellet in 25 ul PBS, add 25 ul 2x SDS Dye and boil. From this, I load 5 ul. Sometimes, I have to play with this a bit (2.5 ul - 10 ul) since the bacteria are at different densities when I harvest.
Attached is an example gel using my method (5 ul each of crude).
Posted 29 July 2009 - 08:05 AM
Did you run a size standard, and if so, did it look good?
Posted 29 July 2009 - 04:51 PM
Posted 30 July 2009 - 12:39 AM
Roo, yours is a really nice picture...
Can overloading be the only reason? In the last lane of the gel, there is a low-concentration sample loaded, that too hasn't ran very well! Is it correct if I say that at least lower bands (which show relatively lower intensity) should have been clear (although little hazy) even if it is a case of high loading?
Posted 30 July 2009 - 06:05 AM
Edited by ram, 30 July 2009 - 06:06 AM.
Posted 30 July 2009 - 06:13 AM
Is this a precast gel or one that you made? I tried doing the 2nd dimension of a 2D-PAGE on a precast gel that was past the expiration date, and it looked horrible.
Posted 30 July 2009 - 06:46 AM
Posted 30 July 2009 - 07:06 AM