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HTRF Human Total CDK7 Detection Kit, 10,000 Assay Points

This lysate is a component of the Total CDK7 kit. It may be used as a positive control for CDK7 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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Feature Specification
Application Cell Signaling
Sample Volume 16 µL

This lysate is a component of the Total CDK7 kit. It may be used as a positive control for CDK7 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 #:
64CDK7TPEG
List Price
USD 2,147.00
Unit Size: 10,000 Assay Points
Part #:
64CDK7TPEH
List Price
USD 12,490.00

Overview

The Total CDK7 cellular assay monitors CDK7 protein levels.

Cyclin-dependent kinase 7 (CDK7) has dual functions in cells: CDK-activating kinase (CAK) that regulates cell cycle progression by phosphorylation of CDK1, CDK2, and CDK4/6, and is also a component of transcription factor II H (TFIIH) that regulates transcriptional initiation through phosphorylation of the RNA polymerase II C-terminal domain (CTD).

Therefore, inhibition of CDK7 has good potential for restoring a controlled cell cycle through the regulation of multiple cell cycle checkpoints, and has emerged as a new potential cancer therapy through cancer specific transcriptional regulation.

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
Technology
TR-FRET
Therapeutic Area
Oncology & Inflammation
Unit Size
10,000 Assay Points

Video gallery

How it works

Total CDK7 assay principle

The Total CDK7 assay quantifies the expression level of CDK7 in a cell lysate. Unlike Western Blot, the assay is entirely plate-based and does not require gels, electrophoresis, or transfer. The Total CDK7 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 CDK7 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.

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Total CDK7 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 CDK7 HTRF detection reagents. This protocol enables the cells' viability and confluence to be monitored.

human-total-cdk7-detection-kit

 

Total CDK7 one-plate assay protocol

Detection of Total CDK7 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-total-cdk7-detection-kit

 

Assay validation

Validation of the specificity of Total CDK7 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 CDK7 detection antibodies. An additional 4 µL of lysates (suplemented 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 CDK7 siRNA led to a 73% signal decrease compared to the cells transfected with the negative siRNA. No signal decrease was observed for cells transfected with CDK1, CDK2, CDK4, CDK6, CDK9, and CDK12 siRNAs. The level of GAPDH measured remained unchanged under all the conditions tested. The data demonstrates that the HTRF Total CDK7 assay is specific for the detection of CDK7 protein and does not cross-react with other CDK family members.

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human-total-cdk7-detection-kit

 

Assessment of total CDK7 levels in human cell lines

Various human cell lines, either adherent HeLa, MCF7, or SHSY5Y cells, or suspension, such as K562 cells, were seeded at 100,000 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 CDK7 detection reagents. The HTRF signal was recorded after an overnight incubation.

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HTRF total CDK7 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 CDK7 detection reagents. Equal amounts of lysates were used for a side by side comparison between HTRF and Western Blot.

Using the HTRF Total CDK7 assay, 1250 cells/well were enough to detect a significant signal, while 5,000 cells were needed using Western Blot with an ECL detection. Therefore in these conditions, the HTRF Total CDK7 assay is 4 times more sensitive than the Western Blot technique.

human-total-cdk7-detection-kit

 

Simplified pathway

Role of CDK7 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).

CDK7 (cyclin dependant kinase 7 ) is both a CDK-activating kinase (CAK), which phosphorylates cell-cycle CDKs (CDK1-CDK2-CDK4 and CDK6), and a component of the general transcription factor TFIIH, which regulates the initiation of the transcription through phosphorylation of the C-terminal domain (CTD) of the RNA Pol II.

human-total-cdk7-detection-kit

 

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HTRF solutions, guide to major applications

This guide provides you an overview of HTRF applications in several therapeutic areas.

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