I need to generate a standard for my pTDP43 MSD assay and an option is to phosphorylate myself a recombinant TDP43 protein with Casein Kinase-1δ. However, I haven't found a protocol to do so. I someone can share a protocol with me it will be really appreciated.
Thank you very much.
Here is a paper that seems to have what you are asking for : https://doi.org/10.1371/journal.pone.0177834. You can substitute the immunopreciptated protein in their protocol for your recombinant one.
I havent worked with Casein Kinase 1δ, but I have done sucessful kinase assays with recombinant CDK like protein kinases. The conditions I use are as follows: 10x kinase reaction buffer (1 ml) : 400 mM Tris-HCl pH 7.5, 100 mM MgCl2, 1 mM EGTA, 10 mM DTT and 1% phosphatase inhibitor (I use Sodium Orthovanadate). Some kinases require 2mM MnCl2. Components of each kinase reaction : 10x reaction buffer 3 μl, Kinase (1ug) 1 μl, Substrate (same molar ratio as kinase) 1 μl, Deionized H2O 22.7 μl, ATP mix 2.3 μl. Total 30 μl.
Good luck.
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