PKC theta - (Oct/29/2014 )

Hello everybody,

I'm trying to detect PKC theta on western blot, the paradox is that I can detect preatty well the phosphorylated form (Thr 538, intact and cleaved form), but not the total unphosphorylated form.....

Does somebody has some suggestion?

XXXXXX

are you using an antibody for the unphosphorylated form or for an epitope which is not phosphorylated?

if you are using an antibody to the phosphorylated form then it may not recognize the unphosphorylated form.

Actually when I use an Ab against the phosphorylated form I see the bands (Uncutted and cut), but when I use an Ab against the total form I just see many bands...........

if you're seeing many bands with the antibody against the total form then you either have several forms and fragments of the pkc or/and are seeing non-specific binding of the antibody.

non-specific binding can be reduced by: reducing concentration of antibody (greater dilution factor), adding or increasing the concentration of tween-20, increasing salt concentration (up to ~0.5M).

you can download a very useful manual about western blotting (as well as others) from this webpage of gelifesciences.

you can also download a very (more) useful manual from millipore: protein blotting handbook

and they also have an online manual for western blotting protocols.