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

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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

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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

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Hi, everyone

I am having the same problem.
Could you solve it?
If so, would you please let us know?

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Hi,

have you tried to block with BSA, Slim Fast or gelantine? I changed from dry milk powder to BSA for detection of phosphorylated proteins, worked a lot better and reduced the background.

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In my experience, blocking with 5% nonfat milk decreases background better than blocking with BSA. Nonfat milk is also cheaper. However, if nonfat milk is not working for you, then try 5% BSA.

In crude lysates, it is common to get background immunostaining of non-specific bands. Try reducing the amount of anti-His antibody.

Also, be sure to wash you blot with a releatively large volume of PBS/0.1% Tween. You should be washing with at least 150 mL-200 mL of PBS/0.1% Tween each time.

Hope this helps.

Good Luck!

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You could also try testing your transfection by Immunoprecipitating the molecule and then blotting with anti-His.

I had the same problem with a cross reactive anti-GFP antibody. If you western blot after an IP there is less protein and hence less proteins with cross reactive epitopes for the poorly designed antibody to light up on your western.

My supervisor and I sent in western data to the company we purchased the GFP antibody from and we got our money back. Too many companies are getting away with selling poor antibodies! If an antibody works poorly or not at all I think we as scientists should demand a refund.

The IP may be a bit of extra work to perform but it sure cleared up my cross reactivity problem, with a similar problem

Good Luck,

Mountainman

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Good idea, I would just add to that the possiblity that the non-specific background could be coming from the secondary antibody. Back off to 1:20,000 or even 1:50.000 and that may go away.

Good luck with it.

LTR

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which anti-His ab are you using?

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Could you also tell the washing condition?

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