The size of protein X (Phosphorylated and non-phosphorylated) will be pretty much the same on a western. Let's say after primary antibodies incubation of the two separate proteins (Phosphorylated and non-phosphorylated) on the membrane; can you pick specific secondary antibodies (one for Phosphorylated and one for non-phosphorylated) that you can in theory detect both bands (Phosphorylated and non-phosphorylated) on the same membrane.
Probably using secondaries antibodies detected with IR fluorescence.
Anyone ever tried this approach?
Jack Kelly , How about urea-PAGE instead of SDS-PAGE, if your protein is small, around 20-30 kDa? Urea-PAGE separation is affected by electric charge and phosphorylated proteins will appear below unphosphorylated proteins.
The best approach to solve this problem is to use phospho specific antibodies if it is available in the market for your protein of interest. The other thing you can interpret from ur blot is that look for some smaller bands around your protein of interest because multiple phosphorylation gives little shift to protein and your primary antibody will detect both the forms (phosphorylated and nonphosphorylate) .
The antibody that recognizes the unphosphorylated protein will probably also recognize the phosphorylated protein, unless it was designed not to do so.
Adam B Shapiro and Poonam Pandey
Thank you for your answers: let me clarify:
- I have antibodies for the phosphorylated and unphosphorylated protein X. So, anti-X and Anti-p-X. These are from different species, Rabbit and mouse.
Can I use anti-X and anti-p-X in the blot without stripping? Since I can detect using different secondaries, my rational is that I can. The same band, I will see protein X with one IR secondary antibody channel; and p-X on another IR channel?
Is there a reason of concern; of having Anti-X and/or Anti-p-X detected simultaneously?
If the anti-X antibody also recognizes the anti-p-X band, it will be in competition with the anti-p-X antibody if both antibodies are used at the same time. The antibodies do not necessarily have the same affinity for their target proteins. If the anti-X antibody recognizes p-X with greater affinity than the anti-p-X antibody does, and you do not adjust the antibody concentrations accordingly to account for this difference, the anti-X antibody may block binding of the anti-p-X antibody to p-X. In that case, you will not detect binding of the anti-p-X antibody to p-X.
- Badges
- Science topic
- Similar topics
- Engineering
- Chemical Engineering
- Membranes