How to get rid of non-specific bands on Western blots. - (Jul/17/2007 )

Hi,

I am trying to run westerns for my protein, and although the antibody that we have detects the protein there are about 7-8 other bands, some more intense than the others on the blot.

We are using post-bleed serum from rabbits. The antibody was procured from Covance. (I was wondering if anyone had any good/bad experiences with them??) Regardless... They have also supplied the Pre-immunization serum, and all the other non-specific bands that I see in my westerns show up in the Pre-immune serum blot. I am using the straight serum that they have supplied to run my westerns.

Could the problem be solved if I purify the antibody? Is there any other way to get rid of the non-specific interactions?
I am using 5% non-fat milk with 0.1% tween for 1hr at room temp to block the blot. Should I switch to some other blocking buffer/blocking conditions?

Thanks in advance for any helpful suggestions.

Vik.

I need a couple of details: is the protein obtained from a bacterial source? Is it overexpressed in E. coli? Does it have a tag: his tag, GST tag? I may be able to help you if you provide that info...

Hey ger225, thanks!!
To answer your questions..
1). It is a chicken protein, and I am running my westerns on chicken tissues homogenized in protein extraction buffer.
2). It has no tags, to the best of my knowledge no post-translational modifications either.

I have tried to attach a scan of the developed film, but there seems to be some problem, the file isnt getting uploaded....
The band that I am interested in, is clearly missing in the pre-bleed blot and is present in the production bleed one.

Covance synthesised the peptide, immunized the rabbits, and gave us the serum of the rabbits, and I have been using straight serum; I havent purified anything.

So I am not certain if the non-specific might be due inefficient blocking, or because the original rabbit antibodies are recognise some chicken proteins. Can I specifically purify my protein of interest... maybe attach the immunizing peptide to some beads, and try and affinity purify the antibody from the serum? What sort of yields could I get, since we dont have a whole lot of serum either!!

I really appreciate any suggestions you could give.

Thanks,

Vik.

It doesn´t sound like inefficient blocking, it seems that the rabbit antibodies recognise chicken proteins. If that is the case, the protocl you suggest for antibody purification is interesting but it seems kinda time consuming... In these cases, it´s better just to focus in your protein of interest and forget about the rest. That´s why in papers, they only show you the band of interest and not the whole Western. Could you do that or is it very important for you to have a one-band-only western?

You could try using 5% BSA in TBS/T, but I'm not sure it would make a difference. You don't say want dilution you use your serum at. Have you tried diluting your serum more in your blocking buffer? I'm not sure it would help but maybe you could reduce the non-specific background.

Ceri

Adjusting the dilution so that you can see your band and reduce the others is a great idea... After that, you just may have to play with the contrast and brightness of your scanned western...

Thanks guys!!

I have tried different dilutions of my antibody, but the problem is what I am calling non-specific bands, are almost as bright, or brighter, depending on what tissue in the chick I am looking at, as my band of interest!!! So, if I dilute the antibody, I sort of loose the resolution on the specific band as well!!!!
Also, we were planning on using this antibody for some Immuno-cyto chemistry... and with the sort of westerns that I have been getting, it sure cant be used for ICC's. So I was wondering if there was some way I could purify the antibody!!
But yes, maybe it will be worthwhile trying a different blocking buffer, and different dilutions in that buffer. Oh yes, I do use my serum at 1:750 dilution!! Is that too concentrated to begin with?? I guess one expects greater efficacy in the purified samples right!! Please correct me if I am wrong, but I am assuming that 1:500-1:750 of the serum is almost the same as maybe a 1:5000 dilution of the purified antibody??!!

and what about a dilution of secondary antibody ?

secondary I use at 1:750 to 1:1000.
would it be useful to try to play around with the secondary too??

depends of secondary provider, but usually i use 1:1000 for rat 1:2000 for mouse and goat, and 1:5000 for rabbit.
reducing secondary reduce background and unspecific... may work but not 100%sure