Why do I get non-specific bands? - with my secondary antibody.... (Sep/26/2008 )

Hi all,

I have done this westerns for like months now... After I load my proteins (not commercial ones, I transfect my protein and doa trizol protein extraction) and also my control peptides (commercial), I block for an hour and then add my primary 1:10,000 dilution and my secondary i used was 1:20,000 dilution. I got too many non specific bands. Then i tried an antibody control and its the secondary thats the culprit. I bound to everything!! Even after over night blocking with 5% milk solution. Today I tried 1:80,000 dilution for my secondary and still go those non specific bands. Can anyone suggest what r the reasons this would happen!! I know it takes a while to optimise antibody concentrations, I am going to do 1:100,000 and 1:200,000 as well, in the meanwhile it ouwld be nice to get some expert opinion on this...

I appreciate any help rendered. Thanks all.

P.S: I also run protein extracted from non-transfected control cells as well and the bands appear for them as well... and I can never see the bands for my control peptides (commercial ones)... Please help!!

Hi,

I tried Western Blot with Trizol isolated protein as well and it does not work properly. The quality of the protein isolate is always bad. i switched to Cytobuster mammalian protein extract buffer (Novagen) and now it is a lot better. I think Trizol is just not really made for protein isolation although they say it.

Stardust

Perhaps you should try to borrow some secondary antibody from a different lab to see if the bands will disappear - if the problem is specific to your secondary. If they do, then order new secondary (secondary antibodies are not that expensive).

Might seem like a stupid question but... when you say you did an antibody control what did you do? Run out lysate and probe the membrane with only secondary antibody (no primary)? If you are getting bands with just this, your secondary needs to go. As smu2 said, borrow a small amount from as many labs as you can to see if one is better. Otherwise, try proteinA-HRP as your secondary. It's not as sensitive but it's a good way to get around some problems. Otherwise you may have to play with washing conditions. Add salt and detergent to try to get rid of non-specific bands.

Big problem though: your antibody never recognizes the purified, commercially bought protein??? Is this what you mean by control peptide or are you running what is supposed to be a blocking peptide? A blocking peptide is usually smaller than the full-length and you might be running it off the gel. What about your transfected cell lysate? Do you see a specific band for the expressed protein? If not your primary might not be specific either (lemme guess...santa cruz). What about the species of protein you are expressing? Are you sure your antibody is recognizing the specific species you are working with?

rkay447 to my rescue again!!!!!!!!!!!!!!!!!!!!!1

Yes, by antibody control i add just secondary antibody and still get the bands..

and my peptide control is my positive control - I shud see bands in this column!!!

And for the rest of ur questions all i can do is

Maybe I shud try a different ptn extraction and also borrow secondaries from diff labs and try to optimise the concentration...

For my transfected lysates, there r loads of proteins there and I see the right size bands when I do a coomasie staining... Because of such non specific bands I am not able to find out if my primary binds to the protein or not!!! so for starters I just want to get rid of the problems with the secondary and then see wat else come up

Thanks for your suggestions guys.. will keep my results posted, in case anybody else out there has a similar problem they can see the suggestions here and learn...

Thanks all!!!

rkay447 to my rescue again!!!!!!!!!!!!!!!!!!!!!1