Dear friends;
I have monoclonal CTL2 antibody. And it is known that CTL2 protein has only 2 isoforms thar are only ~5 aminoacid changed. Bu after 14h incubation at +4 with primary antibody, I get results like my antibody is polyclonal during western blotting
And these bands cant be CTL2 isoforms because As far as I am concerned There shouldnt be that much.
7th band on the marker is my protein.
I tried 1:1000 dilution and even 1:2000 dilution.
You probably need to block with higher milk (or BSA) concentrations and increase blocking time. Sometimes it also helps to use PBS-T instead of TBS-T and vice versa. Extending washing steps can also be helpful. Or your AB does detect more antigenes than the manufacturer tells you.
Well thanks for your answer, we always use %5 dry Milk 1h room temperature. And we never had a problem like these non spesific bands. And there is no bad blocking dots. More over ı wash my membrane 5 Times, each 6 min.
If you incubated for a long time (14h), your got non-specific band. Just 60-90 min is enough for antigen-antibody complex.
you should try blocking with 10% milk, Use a buffer wash with high percent of tween and maybe higher dilution of secondary antibody
Thanks for your answers.
@Kanaporn Poltep , I am not doing the experiment in room temperature. I use my secondery antibody in room temperature for 1 h. When I use my primary antibody in cold room +4 degree for 1 h I see no bands.
@Oscar Badillo I used to try dilution of secondary antibody too, I get the same result with lower band intensity. But still non spesifics.
And I dont really think that blocking is the problem. We use same blotting condition in our lab for all proteins and we never get a non spesific bands with that blotting. More over if I had any non spesific dot, yes blotting could be the problem. But I have non spesific bands, my primary antibody binds to proteins non spesificly. Blotting can not reduce it.
I will try to use a gradient 1:1000 to 1:5000 for primary antibody. I will share the result. Maybe the primary antibody dilution will fix the problem.
1:3000 and 1:5000 dilutions. In 1:5000 I lose my own protein's band density. But still 2 un spesific high density bands.
Dear Enes,
do you see any bands with the secondary only?
Besides this, there could be posttranslational modifications to your protein (ubiquitinylation etc) or results of physiological protease action.
If you have access to the immunogenic sequence or information about the paratope, you also might check possible cross-reactivity with other proteins.
And finally, if you clearly can identify which band represents your target protein, just ignore the rest or have them identified by mass spec sequencing.
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