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Western Blot Forum Discuss western blotting and immunoblot in the western blot forum discussion board. Ask Questions about transfers, blocking, membrane antibody incubation and exposures.


Extra Western bands

Extra Western bands - Western Blot Forum

Extra Western bands - Discuss western blotting and immunoblot in the western blot forum discussion board. Ask Questions about transfers, blocking, membrane antibody incubation and exposures.


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Old 08-12-2010, 02:55 PM
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Question Extra Western bands



After getting no bands on my Westerns, I now have too many! I have bands both bigger and smaller than the predicted size of my protein. I was looking through old posts and saw this is not an uncommon problem - and one old post suggested adding urea to the loading buffer to reduce dimerisation.
So, how much urea exactly do I add? And will any urea do? And why does it work?

Also, I saw several replies suggesting longer blocking - but would this not reduce all antibody binding? I know it's called non-specific binding but the antibody must be recognizing something in order to bind to the protein.

Tatiana
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Old 08-17-2010, 04:20 AM
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Default Re: Extra Western bands

Cross-reactivity and background ridden results are common when optimizing a new antibody. You've given no detail to troubleshoot from so I'll only address the factors that you raised.

Urea may help but the problem is as/more likely to be cross-reactive proteins. Have you checked the published results for evidence that this protein is prone to dimerization? You can use up to 8M Urea, some people advocate changing the SDS-PAGE chemistry, don't heat buffers with urea in them. There are plenty of protocols around and most explain the chemistry briefly. Degassing the gel solution prior to polymerization may help if the issue is dimers.

Changing the blocking agent (casein, BSA, milk powder), percentage of the block, or blocking conditions (temperature or duration) can help. It's possible to over-block but unless you're well outside the normal limits or have a very weak antibody it's unlikely to be an issue.

However, changing membrane (nitrocellulose is often a bit less noisy), and reducing the concentration of primary and or secondary antibodies would be my starting point. I use the same blocking conditions for most antibodies but use a wide range of primary dilutions, temperatures and durations for the primary incubations.

With a new antibody I try to buy antibodies that have been used by multiple labgroups with multiple conditions and then I'll replicate a nice looking published result with the antibody that I'm optimizing. I choose something that easy to replicate with the reagents and lysates that I have. When necessary the tweaking happens after that; first titrate antibody.

That always provides a good base to develop from.
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