How to detect protein kinase from lysate - (Jan/17/2007 )

I wanna ask how to detect protein kinase(Rho-kinase) from cell lysate..
We always use the phosphorylation reaction(Rho-kinase substrate peptide and Lysate) and MASS to detect the level
of Rho-kinase.But the result is not good.
Is there any other method?
Please tell me..thank you..

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what is MASS?

detecting specific kinases in lysates is not easy as there are many other kinases, also phosphatases and many regulators; try immunoblots for unphosphorylated Rho kinase with an appropriate Ab, and if available, use also an anti-phospho Rho kinase Ab for the cisphosphorylated form; if results are still poor do an IP before immunoblotting

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Thank you for your kind reply..
I am sorry...MASS is MALDI-TOF MS.
If i had detected the Rho-kinase level in control cell lines and stimulated cell lines..
are there any ways to detect wheter the transfected polymer containing Rho-kinase substrate(peptide) is phophorylated or not..
Thanks..

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as I understand, you transfect and express the substrate of Rho-Kinase; or there is no need to express as the whole peptide is transfected? it depends on the size of the peptide; you can do it by SDS-Page, for peptides tricine gels are prefered, blot and check with an anti-phospho whatever Aa Ab; you can also work with 33- or 32-PO4 (not recommended); there are advanced methods by FRET with peptides carrying two fluorochromes (YFP-CFP)

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Thanks, Bearer.
We want to transfect DNA&cationic polymer containing Rho kinase substrate peptide complex,
and detect wheter the pDNA is released from the complex due to the phosphorylation of the substrate peptide.
I have tried electrophoresis and HVJ envelope to do the transfection..
but the result is bad.Maybe the selection of cell lines is bad..
or maybe the kinase level is low or something else..

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