Summary
MilliporeSigma 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) solutions |
Remarks: 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-step | 3-step |
|---|
| Screening throughput |
Low to high |
Medium to high |
| Recommended plate types |
384-well |
384- and 1536-well |
| Recommended volume |
20 µl |
6 - 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) |
MilliporeSigma 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) |
| 14-604 |
PI 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) |
| 14-789 |
PI 3-Kinase (p110γ/p85α) |
