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Uneven beta actin

western blot methods

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2 replies to this topic

#1 asta6801

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Posted 15 April 2013 - 05:52 PM

Hi there

I haven't been able to get even beta actin bands. I have had other, much more experienced people in my lab who have no issues like this load my samples and they still get uneven bands too. This leads me to think that it's something other than my loading which is causing the issues. I don't think that it's my samples, because the band intensity of one sample might be low on one gel, but the same sample on another gel might be really intense. Does anyone have any suggestions? I've included two images, they are the same samples run on two different gels so you can see how they look. Picture1.jpg The gels are 8%, I run at 80v for about 2 hours and I transfer them overnight at 30v onto pvdf membranes. I block with 5% skim milk, incubate with primary at 1:10000 for 2 hours, wash, do secondary for 2 hours and then visualise using our chemidoc system.

Thanks!

#2 Tom M

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Posted 17 April 2013 - 12:33 PM

My first thought is that your protein levels are different for each sample. My second thought is that your transfer is not even across the gel, being stronger on one side of the membrane than on the other.

#3 asta6801

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Posted 30 April 2013 - 04:02 PM

Just an update if anyone has similar problems...
I figured out that the results I was getting was because I hadn't sonicated the samples properly.

Thanks





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