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HTRF Human & Mouse Total CDK9 Detection Kit, 10,000 Assay Points

This lysate is a component of the Total CDK9 kit. It may be used as a positive control for CDK9 protein quantification.

For research use only. Not for use in diagnostic procedures. All products to be used in accordance with applicable laws and regulations including without limitation, consumption and disposal requirements under European REACH regulations (EC 1907/2006).

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Offer valid until 12/15. Terms and conditions apply.
Feature Specification
Application Cell Signaling
Sample Volume 16 µL

This lysate is a component of the Total CDK9 kit. It may be used as a positive control for CDK9 protein quantification.

For research use only. Not for use in diagnostic procedures. All products to be used in accordance with applicable laws and regulations including without limitation, consumption and disposal requirements under European REACH regulations (EC 1907/2006).

Click to copy promo code to clipboard.
SAVE 15% NOW on online orders. Use promo code below.
YEAREND15
Offer valid until 12/15. Terms and conditions apply.
Product Variants
Unit Size: 500 Assay Points
Part #:
64CDK9TPEG
List Price
USD 2,147.00
Unit Size: 10,000 Assay Points
Part #:
64CDK9TPEH
List Price
USD 12,490.00

Overview

The Total CDK9 cellular assay monitors CDK9 protein levels.

CDK9 in association with cyclins T or K, also called the positive transcription elongation factor (P-TEFb), is a general transcription factor that regulates transcription elongation through phosphorylation of the C-terminal tail domain (CTD) of RNA polymerase II (RNAP II). This crucial function and the evidence that transcriptional programs are dysregulated in cancer made make CDK9 an attractive target for anti-cancer therapies.

Specifications

Application
Cell Signaling
Automation Compatible
Yes
Brand
HTRF
Detection Modality
HTRF
Lysis Buffer Compatibility
Lysis Buffer 1
Lysis Buffer 4
Molecular Modification
Total
Product Group
Kit
Sample Volume
16 µL
Shipping Conditions
Shipped in Dry Ice
Target Class
Phosphoproteins
Target Species
Human
Mouse
Technology
TR-FRET
Therapeutic Area
Oncology & Inflammation
Unit Size
10,000 Assay Points

Video gallery

How it works

Total CDK9 assay principle

The Total CDK9 assay quantifies the expression level of CDK9 in a cell lysate. Unlike Western Blot, the assay is entirely plate-based and does not require gels, electrophoresis, or transfer. The Total CDK9 assay uses two labeled antibodies, one coupled to a donor fluorophore and the other to an acceptor. Both antibodies are highly specific for a distinct epitope on the protein. In the presence of CDK9 in a cell extract, the addition of these conjugates brings the donor fluorophore into close proximity with the acceptor and thereby generates a FRET signal. Its intensity is directly proportional to the concentration of the protein present in the sample, and provides a means of assessing the protein's expression under a no-wash assay format.

human-mouse-total-CDK9-detection-kit

 

Total CDK9 two-plate assay protocol

The two-plate protocol involves culturing cells in a 96-well plate before lysis, then transferring lysates into a 384-well low volume detection plate before the addition of Total CDK9 HTRF detection reagents. This protocol enables the cells' viability and confluence to be monitored.

human-mouse-total-CDK9-detection-kit

 

Total CDK9 one-plate assay protocol

Detection of Total CDK9 with HTRF reagents can be performed in a single plate used for culturing, stimulation, and lysis. No washing steps are required. This HTS designed protocol enables miniaturization while maintaining robust HTRF quality.

human-mouse-total-CDK9-detection-kit

 

Assay validation

Validation of the specificity of Total CDK9 assay by siRNA experiments

HeLa cells were plated in 96-well plates (10,000 cells/well) and cultured for 24h. The cells were then transfected with siRNAs specific for CDK1, CDK2, CDK4, CDK6, CDK7, CDK9, or CDK12, as well as with a negative control siRNA. After a 48h incubation, the cells were lyzed and 16 µL of lysates were transferred into a 384-well low volume white microplate before the addition of 4 µL of the HTRF Total CDK9 detection antibodies. An additional 4 µL of lysates (supplemented with 12 µL diluent #11) was also transferred into the microplate to monitor GAPDH level using the GAPDH Housekeeping Cellular Kit (64GAPDHPET/G/H) . HTRF signals for both kits were recorded after an overnight incubation.

Cell transfection with CDK9 siRNA led to a 68% signal decrease compared to the cells transfected with the negative siRNA. No signal decrease was observed with cells transfected with CDK1, CDK2, CDK4, CDK6, CDK7, and CDK12 siRNAs. The level of GAPDH measured remained unchanged under all the conditions tested. The data demonstrate that the HTRF Total CDK9 is specific for the detection of CDK9 protein and does not cross-react with other CDK family members.

human-mouse-total-CDK9-detection-kit

 

human-mouse-total-CDK9-detection-kit

 

Assessment of total CDK9 levels in various cell lines

Various cell lines, either adherent HeLa, MCF7, SHSY5Y, NIH-3T3 (mouse) cells or suspension, such as K562 cells, were seeded at 50,000 cells / well in a 96-well microplate. After a 24H incubation, the cells were lyzed with supplemented lysis buffer, and 16 µL of lysate were transferred into a 384-well low volume white microplate before the addition of 4 µL of the HTRF Total CDK9 detection reagents. The HTRF signal was recorded after an overnight incubation.

human-mouse-total-CDK9-detection-kit

 

Total CDK9 degradation using CDK9 Protac

HeLa cells were seeded in a 96-well culture-treated plate under 50,000 cells / well in complete culture medium, and incubated overnight at 37 ° C, 5% CO2. The cells were then treated with Vehicle (DMSO), CDK9 Protac (Thal SNS-032), CDK9 Binder (SNS-032), CRBN Binder (Thalidomide), or an Irrelevant Protac at 1 µM for 4H at 37°C, 5% C02. After cell lysis with 50 µL of supplemented lysis buffer #1, the same lysate was sequentially dispensed into a low volume detection white plate: 16 µL for Total CDK9 and an additional 4 µL (supplemented with 12 µL diluent #11) to check GAPDH levels using the HTRF GAPDH Housekeeping kit (64GAPDHPET/G/H). The corresponding kit detection reagents were added under 4 µL, and HTRF signals were recorded after an overnight incubation. The CDK9 Protac (Thal-SNS-032) induced CDK9 protein degradation with a 84% signal decrease compared to the cells treated with Vehicle (DMSO). No signal decrease was observed for cells treated with CDK9 Binder, CRBN Binder, or an Irrelevant Protac. The level of GAPDH measured remained unchanged under all the conditions tested (no cytotoxicity).

human-mouse-total-CDK9-detection-kit

 

human-mouse-total-CDK9-detection-kit

 

Total CDK9 degradation on HeLa and MCF7 cells

HeLa and MCF7 cells were seeded in a 96-well culture-treated plate under 50,000 cells / well in complete culture medium, and incubated overnight at 37 ° C, 5% CO2.The cells were then treated with increasing concentrations of CDK9 Protac (Thal-SNS-032) for 4H at 37°C-5%CO2. After cell lysis with 50 µL of supplemented lysis buffer #1, the same lysate was sequentially dispensed into a low volume detection white plate: 16 µL for Total CDK9 and an additional 4 µL (supplemented with 12 µL diluent #11) to check GAPDH levels using the HTRF GAPDH Housekeeping kit (64GAPDHPET/G/H). The corresponding kit detection reagents were added under 4 µL, and the HTRF signals were recorded after an overnight incubation.

As expected, the results obtained show a dose-response degradation of Total CDK9 in HeLa and MCF7 cells after treatment with CDK9 Protac (half maximal degradation of 166 nM and 60 nM respectively), while the level of GAPDH remained constant.

human-mouse-total-CDK9-detection-kit

 

human-mouse-total-CDK9-detection-kit

 

HTRF total CDK9 assay compared to Western Blot

HeLa cells were cultured in a T175 flask in complete culture medium at 37°C-5% CO2. After a 48h incubation, the cells were lysed with 3 mL of supplemented lysis buffer #1 (1X) for 30 minutes at RT under gentle shaking.

Serial dilutions of the cell lysate were performed using supplemented lysis buffer, and 16 µL of each dilution were transferred into a low volume white microplate before the addition of 4 µL of HTRF total CDK9 detection reagents. Equal amounts of lysates were used for a side by side comparison between HTRF and Western Blot.

Using the HTRF total CDK9 assay, 300 cells/well were enough to detect a significant signal, while 10,000 cells were needed using Western Blot with an ECL detection. Therefore in these conditions, the HTRF total CDK9 assay is 32 times more sensitive than the Western Blot technique.

human-mouse-total-CDK9-detection-kit

 

Simplified pathway

Role of CDK9 in the cell-division cycle

CDKs are traditionally separated into 2 subfamily members: CDKs that coordinate cell cycle progression (CDK1, CDK2, CDK4, and CDK6) and transcriptional CDKS (CDK7, CDK8, CDK9, CDK12, and CDK13).

CDK9 (cyclin dependant kinase 9) in association with cyclin T or K is a component of the multiprotein complex TAK/P-TEFb, which regulates transcription elongation through phosphorylation of the C-terminal tail domain (CTD) of RNA polymerase II (RNAP II). CDK9 is a key player in the production of mature RNA.

human-mouse-total-CDK8-detection-kit

 

Resources

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Application Note
CDK signaling in oncology: assess the effectiveness of a CDK9-targeting PROTAC molecule

CDK9 is an attractive target for cancer therapeutics due to its crucial role in transcription regulation, particularly of short...

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