EMD: Kinase Kit Resource - K-LISA and Activity Assays

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Kinase Kit Resource: K-LISA™ and Activity Assays

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For direct measurement of protein kinase activity

Summary
Calbiochem� offers kits with several different formats for the direct measurement of kinase activity. K-LISA kits measure kinase activity utilizing a technique in which phosphorylation of a target is detected using phospho-specific antibodies (see Figure 5). Kits utilizing Western blots (Cat. No. 124007 and 420115) offer a straightforward method for measuring kinase activity through detection of phosphorylated protein substrates.

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When to use	Use these kits to measure protein kinase activity and determine the effects of activators or inhibitors. The kits vary in sample compatibility, so this should be considered carefully when choosing a kinase activity kit.
Advantages	• Measure kinase activity directly • Direct effects of inhibitors/activators can be measured
Considerations	Cell lysates or partially purified samples should be used with caution because peptide substrates can often be phosphorylated by multiple kinases.
Sample Type	Varies – see table below
Detection	Varies – see table below
Equipment Required	Colorimetric ELISA plate reader or Western blotting equipment

Figure 5. K-LISA™ Schematic.
The sample containing the protein kinase to be assayed is incubated with ATP and the polypeptide or peptide substrate. The K-LISA Checkpoint Activity Kit (Cat. No. CBA020) uses a biotinylated peptide substrate phosphorylated by Chk1 and Chk2. Similarly, the K-LISA Akt Activity Kit (Cat. No. CBA019) uses a peptide substrate phosphorylated by Akt1, Akt2, and Akt3. The K-LISA PTK Screening Kit (Cat. No. 539701) uses three different random polypeptide substrates (EAY, E₄Y, or EY) composed of different molar ratios of glutamic acid (E), alanine (A), and tyrosine (Y). An HRP-antibody conjugate is added (in some kits a detection antibody is also required), and this is followed by addition of the TMB substrate and
detection at 450 nm.

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Figure 6. Rapamycin + FKBP12 Inhibition of mTOR Kinase Activity measured using K-LISA mTOR Activity Kit (Cat. No. CBA055).
FKBP12 + Rapamycin inhibition of mTOR Standard. The activity of the mTOR Standard, (50 ml) was determined in the presence of either Rapamycin (20 mM; Cat. No. 553210 or 553211), Rapamycin (20 mM) + GST-FKBP12 (37 mg/ml), or Wortmannin (10 mM; Cat. No. 681675; also included with the kit). Addition of Wortmannin serves as a positive control for inhibitor analysis. Inhibition profiles can be generated based on mTOR activity in the presence and absence of test inhibitor(s).

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Figure 7. Detection of JNK activity with JNK Activity Immunoassay Kit (Cat. No. 420115).
The assay utilizes a JNK-specific antibody to immunoprecipitate JNK from cell lysates, after which JNK activity is detected in a kinase reaction using c-Jun as a substrate. Phosphorylation of c-Jun can be analyzed by Western blot using a c-Jun phosphospecific antibody. An increase in JNK in Jurkat Cells following anisomycin treatment is shown. NOTE: The heavy chain of the precipitating antibody is detected by the secondary antibody.

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K-LISA Kits

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Activity Assay Kits

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Related Resources � Related Products for K-LISA Kits

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