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Nonspecific Binding on Western Blot


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4 replies to this topic

#1 Ihab

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Posted 02 November 2004 - 11:35 AM

;) Hi Everyone,

I am getting a lot of non-specific bands that show up on my western blot .

I am using an anti-his monoclonal antibody that is verified to give almost no background. I am using a goat anti-mouse polycolonal for my secondary Ab.

When I do the HRP detection, I get a lot of non-specific bands that show up on all samples (including the negative control and positive control) and I can't tell if my protein of interest is one of them or not.

I am blocking with 5% dry milk and incubating the antibodies in blocking buffer as well. I am also washing the membrane pretty good. Any suggestions? thanks

#2 Oleksii

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Posted 03 November 2004 - 10:21 AM

Hi,

What is your sample? Is it lysate from eukaryotic cells? Anti-His Ab can has some non-specific binding with eukaryotic proteins. You can try titrating of loading of your sample. And as well play with your anti-His Ab dilution. Try to find lowest amount of your sample for which you still have a signal, very often non-specific band will disappear at this conditions.

Cheers,

Alexei

#3 Ihab

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Posted 04 November 2004 - 10:49 AM

Yes, I am using lystate from eukaryotic cells. The transfection efficiency in the cell line I am using is usually low, so my protein is probably being expressed at low levels. The western blot shows about non-specific bands in the entire lane and I can't tell if my protein is showing up or not.

Thanks

#4 msochor

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Posted 14 August 2009 - 07:44 AM

I have the same problem in E. Coli. My problem however is due to the presence of a reducing agent in my lysis buffer. I am using Beta-mercaptoethanol (BME) as my reducing agent, which according to this reference http://www.ncbi.nlm..../pubmed/2212467 will cause non-specific binding.

So I guess the lesson is, no BME if you are going to be doing a western. Use DTT instead.

#5 miBunny

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Posted 14 August 2009 - 05:53 PM

try titering down your primary antibody. You can also try incubating the primary at 4 degrees overnight (which can help with background)

Have you tried different blocking agents (5% BSA or 3 % ova)?

Have you tried switching PBS for TBS (or vice versa)?

Do you have tween in your wash and block buffers?




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