Myosine Light chain WB - (Mar/13/2008 )

I'm trying to detect MLC phosphorylation by Western Blot but I can not even detect the total form. I'm using endothelial cells and my lysis buffer is based on RIPA ( Trsi 50 mM, NCl 150 mM, DOC 0,25%, SDS 0,1%, NP40 1%). Papers show beautiful WB with strong signal with the same antibody I'm using.
Can somebody help me to undertsand where I'm wrong?
Thanks

Don't you use phosphatase- and protease-inhibitors? This is essential, as your protein will be degraded otherwise and the phosphorylation signal will get lost. And always keep your samples on ice during lysis for the same reason. How is your blot working? Can you detect a protein like GADPH or actin nicely or is there something wrong during the procedure? Have you checked with ponceau-red for transfer?

Of course there is antiproteases and orthovanadate. I'm doing lysates and WB routinely, so I'm convinced it is something specific to MLC. Lysates are done on ice, lysisbuffer is cold so nothing to do with eventual protein degradation. Blots are OK, ponceau OK, I can detecte on the same blot actin but also others phosphorylated proteins. The point is that I can not detect MLC protein.

you may need an antibody to a different phosphorylated moiety.

as the bearer said you may want to radioactively phosphorylate the mlcs but i would recommend thiophosphorylating (use atp-gamma-35s). the bond is more stable.