Hi! I'd like to assess PKA activity by resolving non-phosphorylated and phosphorylated kemptide on native agarose gel, but the kemptide I have is not fluorescently-labelled. So,
Kemptide is a phosphate acceptor peptide that serves as a synthetic substrate for PKA with the following sequence (7 aminoacids): H - Leu - Arg - Arg - Ala - Ser - Leu - Gly - OH
1) Has anyone tried to stain kemptide with coomassie blue directly on the agarose gel?
2) Would I be able to transfer the agarose gel to a PVDF membrane (western-blot experiment) to develop the membrane using anti-phospho PKA substrates antibody? Is there any protocol or particular considerations I might need?
Thank you!!!!
Cintia,
I think your problem is asking for a competitive ELISA or HPLC to detect and quantify the phosphorylated peptide. A molecular mass of about 1kD can't be resolved well (if at all) on gels, blotted onto a membrane or stained with Coomassie (you'd need quite an amount to see it).
Wolfgang Schechinger Hi Wolfgang! Thank you for your reply :)
I'd agree with you but there are assays were phosphorylated kemptide can be resolved from non-phosphorylated kemptide just fine on 0.8 % native agarose gels. This is possible because the two forms migrate towards different electrodes (The phospho-kemptide has a negative charge and it migrates towards the anode, while non-phospho-kemptide has a positive charge and migrates towards the cathode). However, those assays use fluorescent-labeled kemptide and so detection is carried out using a transilluminator. I only have non-labeled kemptide, and that is why I'm trying to figure out a different detection method.
I have overlooked that, only thought about resolving by size. Is there maybe an opportunity for you to label the peptide with e.g. FITC or with fluorescein and NHS/EDC chemistry (sometimes these reagents are hidden in the back in of a freezer from times when it was common to label proteins oneself).
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