Recently I have been having trouble with my Western Blot protein bands taking a "mickey mouse" shape rather than the usual linear band. In some cases, the shape is even more exaggerated. This does not occur in all lanes on my membrane, which leads me to believe that it is not a problem with running my gel or even with the transfer.
My cells are lysed in RIPA buffer. I save them in 25 microgram aliquots, and store them in the -80. Once I am ready to load the gel (usually several days or weeks later), I thaw my samples on ice, add 6 x SDS sample buffer (1 part buffer to 5 parts sample), and then heat for about 5 minutes. Finally, because my samples are still very viscous at this point, I add 25 units DNase and heat for about 8 minutes. Then I let the samples cool before loading.
I would greatly appreciate feedback from those who have ideas as to how I can improve my protocol and potentially eliminate my problem! Thank you.
LAH603Member Since 05 Oct 2012
Offline Last Active Oct 11 2012 09:30 AM
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16 Oct 2012 - 03:03