EMD: Kinase Kit Resource

Kinase Kit Resource: Technical Tips

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1. What's the best way to get consistent results when using a kit?

Always use components from the same lot of a given kit, including the standard, since each lot contains kit components that are optimized to work together.
When applicable, perform a standard curve with each new experiment.
Use externally prepared standards (e.g., purified enzymes, cell lysates) only as experimental controls; do not replace the included standards with standards from another source.

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2. What types of samples can be used?

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Different kit protocols are optimized for use with different sample types, so it is recommended that this information be confirmed in the protocol. Depending on the enzyme, the level of detectable activity can vary among sample types. It is also important to note any species reactivity limitations.
Using subcellular fractions (e.g., nuclear or membrane fractions) may improve target detection. Try the ProteoExtract� Subcellular Proteome Extraction Kit (Cat. No. 539790) to prepare four separate fractions from one sample: cytoskeletal, nuclear, membrane/organelle, and cytosolic.
For optimizing detection of phosphorylation, use phosphatase inhibitors or PhosphoSafe™ Extraction Reagent (Cat. No. 71296), an enhancement of the CytoBuster™ Protein Extraction reagent, with four phosphatase inhibitors, to prepare your sample.

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3. How do I choose a protein kinase inhibitor for my assay?

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Consider which protein kinase must be inhibited to achieve the experimental results
desired. For a direct kinase assay such as K-LISA, inhibit the kinase being assayed, but for PhosphoDetect™ ELISA kits, choose inhibitors that affect kinase(s) upstream of the target.
Consider cell permeability, mode of action, specificity, and K_i/IC₅₀ values for each inhibitor. Refer to the Inhibitor Sourcebook™ or our online inhibitor resource, to find this information. Refer also to two excellent reviews on kinase inhibitor specificities: Davies, S. P., et al. 2000. Biochem. J. 351, 95; Bain, J., et al. 2003. Biochem. J. 371, 199.
In general, samples containing protein kinases of interest should be pre-incubated with an
inhibitor for 15-30 min prior to performing the activity assay.

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