LAT western blot... 36 and 75 kDa? - (Jun/08/2007 )
Hi,
I am doing a western blot with T stimulated cells and I have got a band arround 40 kDa and a biggest band at 75 kDa. Would it be a dimer? I use a specific phospho antibody against the Tyr226 phosphorylation of LAT and I have multiple bands (Why?), a strong band at around 40 kDa but a stronger band at 75 kDa.
Thanks for your help!
Brassap
Ahh.. the fun of working with antibodies. Can't really explain the multiple bands but most antibodies do have background. Is the 75 kDa band present in your negative control (unstimulated cells)? If so, this is just a background band that can be ignored. Additionally if you look on abcam's website, they have a picture of their Y226-specific antibody and while it's not the prominant band, they too appear to have a slight band around 75. If this is a SDS gel, there is no way this could be a dimer as SDS-PAGE is a denaturing gel. Keep your dilution of antibody.. I've seen dilutions that get better and better with use as the background bands may become diminished or completely absent.
I am doing a western blot with T stimulated cells and I have got a band arround 40 kDa and a biggest band at 75 kDa. Would it be a dimer? I use a specific phospho antibody against the Tyr226 phosphorylation of LAT and I have multiple bands (Why?), a strong band at around 40 kDa but a stronger band at 75 kDa.
Thanks for your help!
Brassap
the p75 could be a dimer of p40; however, it also could be an unspecific reaction as the Ab detects the epitope around phospho Tyr which may not be unique; if available, do in parallel a blot with peptide-blocked Ab, and see of again both bands are detected or not
Thank you very much to both of you for your constructive input!
Have a great day!
Brassap