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Western Blotting


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#1 _Agnes_

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Posted 28 June 2010 - 10:18 AM

Dear all,

I am writing to know your opinion about some strange results I got while performing WB.
#1. Is that possible that different antibodies for the same protein stain at different molecular weights? If yes, why does that happen?
#2. I used RIPA buffer (+ protease inhibitors) to extract samples from mouse tissue. I incubated the membranes (NC) with the ab ON (4C) in 1% milk solution in TBST and, with one of the antibodies I got good results but when I tryed to replicate them in the same conditions I got a lot of extra bands and the more intense band is lower than the correct protein molecular weight. I have no idea about what might be happening and how I can get rid of the extra bands.
All help/ ideas are welcomed.

Kind Regards,


Agnes

#2 macroman

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Posted 28 June 2010 - 12:54 PM

Normally i would like to use 5% Milk in TBST for blocking.

#3 Ahrenhase

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Posted 28 June 2010 - 03:08 PM

I don't think you should get extra bands unless you have degradation, but you said you were using protease inhibitors. Where did you get your antibodies? And what does the rest of your procedure look like?

#4 laurequillo

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Posted 28 June 2010 - 07:56 PM

Normally you use 5% milk or BSA. The extrabands could be that the antibody is not so specific, that you are using too much antibody, loading too much protein...or just different forms of the protein.
Sometimes the antibodies are not as good as they say in the datasheet! ;)
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