Hi,
I'm doing western blot for phospho-RhoA using rat brain tissue extract. But I'm getting double bands around the right molecular weight, and they are very close. Is there any idea of what they might be? Is it possible that the antibody detects PTM of RhoA? And how to test which is the right band in terms of phospho-RhoA?
Thanks a lot!
Hello
The upper band is the right band for phospho-RhoA because phosphorylation of protein causes an upward shift in mobility.
There is a high possibility that the primary antibody may be detecting both phospho-RhoA and non-phospho-RhoA because the molecular weight of phospho-RhoA is 22kD and that of non-phosphorylated RhoA is 21kD. Both are very close to each other.
Best Wishes.
I agree with Malcom Nobre RE the antibody - how certain are you that it is specific for phos-RhoA? (i.e. does the manufacturer provide negative blots of non-phos-RhoA?). Alternatively, RhoA could show multiple phosphorylation profiles or extra PTMs giving two separate populations of phos-RhoA. Especially as it's from tissue extract - there's all sorts of PTMs going on in the brain!
As for identification, LC-MS/MS can identify PTMs and indicate which residue is modified. Although best to take it with as a suggestion, not proof.
You can have also two phosphorylations.
Then you can see the first band with a protein RhoA with one phosphosite (recognized by antibody). And the protein in the second band can be also phosphorylated on the same residue (recognized by antibody) plus additional second phosphorylation.
- Badges
- Science topic