HTRF®Class I PI 3-Kinase Assays

A Guide to Kit Selection


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Summary
EMD Millipore offers robust and sensitive HTRF® Class I PI 3-Kinase kits, available in both 4-step and 3-step formats to optimise assay volume and throughput for all screening requirements.
Configuration of the Class I PI 3-Kinase Kits
4-step (original) 3-step
Step 1: + Reaction Buffer (including kinase and PIP2 substrate) Step 1: + Reaction Buffer (including biotinylated PIP3, kinase and PIP2 substrate)
Step 2: + ATP (to initiate kinase reaction)Step 2: + ATP (to initiate kinase reaction)
Step 3: + Stop solutions (including biotinylated PIP3)Step 3: + Stop/Detection Mix Solutions
Step 4: + Detection Mix solutions
Remarks: In this assay all components of the FRET complex are added at the end of the assay, via the Stop and Detection Mix (DM) solutionsRemarks: In this assay the biotinylated PIP3 is mixed with the reaction buffer and is therefore present from the start of the reaction. This does not affect the activity of the kinase during the assay, but enables the remaining components of Stop solutions A and B (as used in the 4-step kit) to be combined into a single Stop solution, which can be further mixed with the Detection Mix components prior to addition to the assay plate. This feature is not possible with the 4-step kit due to the requirement for the biotinylated PIP3 to equilibrate with the PIP3 reaction product before it is exposed to the GST-tagged PH domain.
Comparison of 4-step and 3-step Assays
The reduction in number of reagent additions makes the 3-step format ideal for high throughput screening. A further advantage of the 3-step configuration is that the total reaction volume can be reduced from 30 µl down to less than 10 µl (typically 6 µl, dependent on the availability of suitable liquid handling equipment). This makes it readily suitable for use with 1536-well plates.

4-step3-step
Screening throughputLow to highMedium to high
Recommended plate types384-well384- and 1536-well
Recommended volume20 µl6 - 20 µl
Reference Inhibitor Data
Titration of the known PI 3-Kinase inhibitor PI-103 demonstrates that the 4-step and 3-step assay formats produce comparable data.





Assays were conducted at 100 µM ATP. Data points represent the mean of three replicates and are expressed in terms of the % kinase activity compared to the DMSO control wells.
Catalogue Number Information
Kits for assaying Class I PI 3-Kinases using either the 4-step or 3-step method are available in various sizes. Please note that the kinase is not included in the kit and should be purchased separately. Screening kits for Class II PI 3-Kinases and PIPKINs are also available.

Cat #Description
33-016 PI 3-Kinase HTRF Assay - 384 wells (4-step)
33-017 PI 3-Kinase HTRF Assay - 1920 wells (4-step)
33-036 PI 3-Kinase HTRF Assay - 10,000 wells (4-step)
33-037 PI 3-Kinase HTRF Assay - 50,000 wells (4-step)
33-040 NEW PI 3-Kinase HTRF Assay - 384 wells (3-step)
33-041 NEW PI 3-Kinase HTRF Assay - 1920 wells (3-step)
33-047 NEW PI 3-Kinase HTRF Assay - 10,000 wells (3-step)


EMD Millipore offers the following Upstate®-brand Class I PI 3-Kinases that are compatible with the kits shown above:
Human enzymes
Cat #Description
14-790 PI 3-Kinase (p110α/p65α)
14-602 PI 3-Kinase (p110α/p85α)
14-782 PI 3-Kinase (p110α (E542K)/p85α)
14-783 PI 3-Kinase (p110α (E545K)/p85α)
14-792 PI 3-Kinase (p110α (H1047R)/p85α)
14-603 PI 3-Kinase (p110β/p85α)
14-558 PI 3-Kinase (p120γ)
14-604PI 3-Kinase (p110δ/p85α)

Mouse enzymes
Cat #Description
14-786 PI 3-Kinase (p110α/p65α)
14-785 PI 3-Kinase (p110α/p85α)
14-791 PI 3-Kinase (p110α(E542K)/p85α)
14-781 PI 3-Kinase (p110α(E545K)/p85α)
14-787 PI 3-Kinase (p110α(H1047R)/p85α)
14-794 PI 3-Kinase (p110β/p85α)
14-788 PI 3-Kinase (p110β/p85β)
14-789PI 3-Kinase (p110γ/p85α)


Please contact us for further information: uk.project.manager@merckgroup.com