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Alpha Kinase Assays


Overview


AlphaScreen® and AlphaLISA® technologies can be used to perform tyrosine, serine/threonine, and lipid kinase assays. Compatible kinase substrates include lipids, peptides, and full length proteins. Substrate capture by the beads is achieved using protein tags (GST, 6-His), chemical labels (biotin, digoxygenin, FITC), or by using specific antibodies to detect unmodified endogenous proteins. The AlphaScreen PhosphoSensor kit allows you to detect substrate phosphorylation when suitable antibodies are unavailable, or when the exact phosphorylation site is unknown. Both AlphaScreen and AlphaLISA technologies can be used for biochemical in vitro assays, though AlphaLISA beads can also be used when working with more-complex matrices such as serum samples or cell lysates. If you are interested in cell-based kinase assays, we recommend that you check the Alpha SureFire® kits list to see if a kit already exists for your target of interest.

There are three major formats for Alpha kinase assays:

  1. Detection of phosphorylated substrate using generic or specific anti-phospho antibodies.
  2. Detection of phosphorylated substrate using our AlphaScreen PhosphoSensor antibody-free assay.
  3. Detection of substrate binding to kinase using an Alpha biomolecular interaction assay.

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Alpha products and catalog numbers


View a listing of Alpha products with catalog numbers.

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General steps for kinase assay development


Optimization steps in brief

  1. Enzyme titration (control, no enzyme)
  2. Kinetic study (time-course)
  3. Substrate titration
  4. Antibody titration (if applicable)
  5. ATP titration
  6. Inhibition curve
  7. Z' experiment (if desired)

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Buffer selection


Visit Buffer selection for Alpha assays for help in choosing a suitable buffer for your assay. This page describes the various Alpha immunoassay buffers supplied by PerkinElmer and will help you avoid interfering substances in buffers of your own design.

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Anti-phospho antibody-based assays


Detailed information on assay development, where to start, tips and what you need to run this assay.

Alpha-anti-phospho-assay 
Figure 1. Alpha kinase assay using an anti-phospho antibody.

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Alpha SureFire® cellular kinase assays


Detailed information on assay development, what you need to run this assay, tips, etc.

SureFire both formats 2 
Figure 2. Alpha SureFire® assay to study cellular phosphorylation events.

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PhosphoSensor assays


Detailed information on assay development, where to start, tips and what you need to run this assay.

AlphaScreen_PhosphoSensor_ASK
Figure 3. Alpha kinase assay using the AlphaScreen PhosphoSensor kit.

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Substrate binding assays


View more information on assay development, what you need to run this assay, tips, etc.

Alpha_substrate_binding_ASK
Figure 4. Alpha kinase substrate binding assay.

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Citations


View a brief list of citations for Alpha kinase assays.

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General tips


  • Information is provided on the pages linked from each assay format described above.
  • Echelon makes a PI3 kinase adapter kit (#K-1300) that works with our AlphaScreen GST detection kit (catalog number 6760603C/M/R) to measure the phosphorylation of PIP2 by PI3 kinase (PI3K). Please see our poster and refer to their website for more information.

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Troubleshooting


View general Alpha troubleshooting tips.

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Custom bead conjugation and custom assay development at PerkinElmer


PerkinElmer offers custom bead conjugation services as well as custom assay development. If you are interested in having your biomolecule custom-conjugated to a bead, or in custom assay development, please contact our custom teams:

ON>POINT® Custom Labeling and Conjugation ServicesON>POINT® Custom Assay Development Services

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