3 replies to this topic

[YVETTE]

I am doing western blot to detect the phosphorylated Jak2 and phosphorylated STAT4. I have tried three times, never succeed. The first antibody was from Cell Signalling. The actin was okay.  
I am wondering if there are any problem with lysis buffer which couldn't extract phosphorylated protein.  I am not skeptic on the antibody, because I was using STAT1-p from the same company which was excllent.
any suggestion on this will be appreciated.

[mdfenko]

are the antibodies certified for western blot?

if not then they may not recognize denatured epitopes.

if they are then, if you have any purified stat4 and/or jak2, you can try a dot blot to check the antibodies (you can check native and denatured).

you should not have solubility problems with the phosphorylated proteins (unless there is calcium present in relatively high concentration).

[YVETTE]

mdfenko, on Mar 10 2010, 01:04 PM, said:

are the antibodies certified for western blot?

if not then they may not recognize denatured epitopes.

if they are then, if you have any purified stat4 and/or jak2, you can try a dot blot to check the antibodies (you can check native and denatured).

you should not have solubility problems with the phosphorylated proteins (unless there is calcium present in relatively high concentration).

Thank you so much for your reply. actually I am not sure if the antibody are certified for WB or not. these are new ordered Ab. can you let me know how to check it. thanks

[mdfenko]

YVETTE, on Mar 10 2010, 06:06 PM, said:

Thank you so much for your reply. actually I am not sure if the antibody are certified for WB or not. these are new ordered Ab. can you let me know how to check it. thanks

the data sheet that came with the antibody should give you the information. it should also be available in the catalog so that you can make the right choice when purchasing antibodies.