double bands - (Dec/03/2008 )

Hi,

I have been obtaining double bands recently in my western blots which I did not find before with the same proteins. Earlier I used to heat the samples at 70 degree C for 10 minutes and now I am doing it at 95 degree C for 5 minutes. Can that be one of the problem!! Couldn't figure out the exact reason. And hey, the primary and secondary antibody concentrations did not change.

Any answer would be of great help to me.

Thanks.

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[Your information is incomplete. What is the expected band size ??? What is the band size of the additional one????
I can say that some times increased protein concentration or antibodies also pops up additional bands.

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Is it possible that your protein exists in a complex of more than one form of itself? Has the primary band size changed since you started using your 95 degree step? if not, then there is may be some cross-reactivity issues with the antibody.

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In addition to the above responses,

The heat conundrum you can easily resolve by treating two samples at 70 and 95'c, and running them side-by-side for western.

You did not mention the original material.

--Are you using the same cells?
--if yes, are they cultured in the same way as earlier? (same media, same culturing time before western, cell-cycle synchronization etc)
--if yes, have you changed the lysis buffer in any way? (i.e. are you adding phosphatase inhibitors lately?)

Answer to these questions is important because the most common natural cause of two bands is post-translational modification (phosphorylation etc). If your cells, or cell treatment, or cell lysis method changes, you may start seeing/not seeing extra bands representing modfied forms of protein.

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