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"Mickey Mouse" bands

Western Blot

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#1 LAH603

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Posted 05 October 2012 - 12:19 PM

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.

#2 bob1

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Posted 05 October 2012 - 01:23 PM

It sounds like it might be a salt problem, but could you post a picture so we can be sure of the effect you are talking about

#3 LAH603

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Posted 08 October 2012 - 07:39 AM

Here is a photo to illustrate what I am describing...

Attached Thumbnails

  • ICAM_30s_high (2).jpg


#4 bob1

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Posted 08 October 2012 - 01:20 PM

Does it occur consistently in the sample(s) that you have seen it in?

Are you boiling the samples for 5 min, or is it a lower temp?

You may be lysing your cells at too high a concentration - what volume is the 25 ug in?

#5 LAH603

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Posted 09 October 2012 - 06:23 AM

It does not happen with all samples, despite the fact that I prepare them in the same manner.
I only heat my samples for five minutes. I do not boil them.
Twenty-five micrograms of protein is usually in a volume of 12-20 microliters.

#6 bob1

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Posted 09 October 2012 - 12:36 PM

Does the odd band formation happen in those samples with lower volume (i.e. higher concentration)?

i suspect that you are seeing some sort of aggregation/complex formation which is resulting in retarded migration. If you can, try boiling for 5 min or heating to 70 C for 10-15 min, one of the samples that consistently does this - this should denature the samples completely in the presence of SDS and a reducing agent.





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