I wanna check the PRDX3 (Peroxiredoxin) and Phosphorylated PRDX3 level in my protein samples, so for that purpose I require antibodies for performing Western blot.
The antibody for PRDX3 is available but I cant find the antibody for phosphorylated Prdx3 (At threonine 146 position).
Is there any other way out besides Western?
plz help.
If you can control the phosphorylation event (either in cell culture or in cell/tissue homogenates), then you can use 32P-ATP to radiolabel the phosphate that is added to your protein. Afterwards you can detect your protein via regular Western blot and the phosphorylated protein via autoradiography.
You can try the Zn-Phos-tag SDS-PAGE technique, which allows separation of forms of proteins with different phosphorylation levels based on their migration on gels. You can run your gel and then do a blot, the antibody will recognise both forms of the proteins but they might (should) have a different migration.
Just to mention it, you could also try to find a general P-Thr antibody which is working for your protein. This might however not be a trivial task, but the answers to a related question found here (https://www.researchgate.net/post/Anyone_know_a_good_anti-phosphothreonine_and_anti-phosphoserine_antibodies_for_IP_and_WB) might be helpful.
You might check out nano-immunoassay, which uses isoelectric focusing to separate out different phosphorylated species and then detects using an antibody against the protein (needs to recognize both P and un-P).
http://www.proteinsimple.com/app_post_translational_modification_characterization.html
http://tasc.stanford.edu/services/nia-overview.html
If your Ab is proper for immunoprecipitation of your target protein, pull it down with your Ab and run the precipated protein in WB. Then probe your membrane with an ordinary anti-phspho-Tyrosine or P-Serine Ab (depends if you are looking for which one).
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