I am using cell signalling primary antibody and secondary antibody from sigma and substrate is NBT/BCIP.
primary Ab dilution 1:1000 (3hrs rocking)
sec Ab 1:10000 (45min rocking)
primary ab dilution buffer -5%BSA+0.1% between 20% and 0.1% sodium Azide
If I am using sigma primary and secondary I won't get multiple band if I changed primary from cell signaling and secondary from sigma I got multiple bands.
I'm attaching the image for reference.
The difference obviously is in the primary antibody, I would say that the Cell Signaling antibody is just not that good (though they often have very good ones). Just stick with the primary from Sigma. Generally, different companies is not a problem, rather the problem lies with a specific antibody. We use all secondary antibodies from DAKO and primary antibodies from several companies includingCell Signaling, Sigma, Abcam, Santa Cruz and others. I have had both good and bad (i.e. multiple bands) primaryantibodiesfrom all of these.
I agree that the problem is the primary. However, what is the band you are trying to detect? Can you see it with both primaries? Even though you get multiple non-specific bands with the Cell Signaling antibody - you may be able to change your blocking conditions to increase the signal to noise. How are you blocking?
Hi Asiya,
Do you expect any isoforms (splice variants) for your protein? Do you have the sequence of the antigens used for immunization by both companies? I would say most chances are that the Cell Signaling primary is recognizing an additional protein, whether this is specific or unspecific depends on tons of different factors.
I may caution about the problem is the primary antibody. But do agree that it depends on whether it is the right band you are expecting or not. The ideal condition is to use blocking peptide that can block out the band to validate the antibody. I have studie nitrated protein and one company with good reputation gave me only one band whereas we knew it should be multiple bands. Some antibody will identify protein with different post-translational modifications and then give out multiple bands on blot which maybe better than just one band.
- Badges
- Science method