I am working with A.thaliana protein. I have a heat sensitive GFP tag. So I cannot heat my samples before loading onto the gel for western blotting.
I Incubated the blot with Rabbit-AntiGFP antibody and goat anti-Rabbit HRP secondary antibody (both from cell signalling) and when developed, I see huge bands at 55kDa rather than expected size of 100kDa.
But the same blot when stripped and reprobed with mouse Anti-GFP (Roche), I see the protein bands at expected size
I dont know why I am unable to see the bands with Rabbit antibodies? Anyone worked before with nonheated protein samples probing please help.
I have attached the picture below for reference
Pic-1: Combnd 1' - Probed with rabbit antiGFP
Pic2: Combnd 3'' - Probed with mouse anti GFP
Hi Chaitra Hiremath
There are several possible explanations for the observed non-specific binding with the rabbit antibody and specific binding with the mouse antibody when using non-heated protein samples in a Western blot:
To help distinguish between these possibilities, you could try the following:
It is important to optimize the Western blot protocol for the specific target and sample type, as different antibodies and detection methods may have varying levels of sensitivity and specificity.
Do you have data without the primary antibody, i.e., the background signals?
Wolfgang Schechinger I did a blot only with secondary antibody and I had no signals at all. So i assume as mentioned by Anjali Srivastava it is the antibody specificity
Anjali Srivastava thank you for your kind reply. I tried different blocking and washing agents and also increased the blocking concentration but in vain. I even tried to contact the antibody vendor and unfortunately they also had no explanation for this problem.
May be i will consider other options mentioned by you and get back with my results.
Thanks again
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