[³³P] Lck kinase enzyme assay

³³P incorporated into biotinylated peptide substrate.

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1536-well assay protocol

1.	3 µl reaction mix containing: 0.02 µCi [g-33P]ATP (AH9968) 0.21 mM ATP 0.42 mM peptide substrate 16 nM Lck enzyme in 50 mM HEPES, pH7.0, 10 mM MgCl2, 1 mM DDT, 0.1% (w/v)BSA
2.	Incubate for 40 minutes at 25oC
3.	3 µl stop buffer containing: 0.1 mg polystyrene streptavidin (RPNQ0261) or yttrium oxide streptavidin (RPNQ0271) bead in 50 mM HEPES, pH7.25, 50 mM EDTA, 0.1% BSA (w/v), 0.1% Triton X-100(v/v), 50 mM ATP
4.	Centrifuge at 130g for 3 minutes
5.	Image for 10 minutes

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Reagent usage in 384- and 1536-well format

Assay components 384-well 1536-well Assay volume 75 µl 6 µl Bead 250 µg 100 µg Enzyme amount 800 fmoles 48 fmoles Substrate 0.42 mM 0.42 mM Substrate amount 20.8 pmoles 1.25 pmoles ATP 0.21 mM 0.21 mM ATP amount 0.4 pmoles 0.625 pmoles [33P]ATP 0.1 µCi 0.02 µCi

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Typical data

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Ordering information

The following products were used in this assay and can be purchased online

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