- In Gel Kinase Assay.
ABSTRACT
Proper spatiotemporal regulation of protein phosphorylation in cells and tissues is required for normal development and homeostasis. We present the protocol 'In Gel Kinase Assay', which is useful for protein kinase activity measurements from crude protein extracts. We have successfully used 'In Gel Kinase Assay' protocol to show that the Arabidopsis thaliana sextuple mutant in the PYRABACTIN RESISTANCE1/PYR1-LIKE/REGULATORY COMPONENTS OF ABA RECEPTORS (PYR/PYL/RCAR-ABA receptors; line pyr/pyl112458) is impaired in ABA-mediated activation of SnRK2.2, SnRK2.3 and OST1/SnRK2.6, as much as the triple mutant snrk2.2/2.3/2.6 (Gonzalez-Guzman et al., 2012).
MATERIALS
Product Number
Brand
Product Description
Sigma-Aldrich
SIGMAFAST™ Protease Inhibitor Cocktail Tablets, EDTA-Free, for use in purification of Histidine-tagged proteins
Sigma-Aldrich
Ethylene glycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid, Molecular Biology, ≥97.0%
Sigma-Aldrich
Ethylenediaminetetraacetic acid disodium salt dihydrate, suitable for electrophoresis, Molecular Biology, 99.0-101.0% (titration)
Sigma-Aldrich
Sodium dodecyl sulfate, BioReagent, suitable for electrophoresis, Molecular Biology, ≥98.5% (GC)
Sigma-Aldrich
β-Glycerophosphate disodium salt hydrate, BioUltra, suitable for cell culture, suitable for plant cell culture, ≥99% (titration)