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HTRF Human Total CDK1 Detection Kit HTRF®

The Total CDK1 kit is designed to monitor the expression level of cellular CDK1 (Cyclin-Dependent Kinase 1).

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The Total CDK1 kit is designed to monitor the expression level of cellular CDK1 (Cyclin-Dependent Kinase 1).

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Overview

CDK1 , also known as Cell Division Cycle 2 (Cdc2), is a serine/threonine kinase that is ubiquitously expressed in eukaryotes. CDK1 is a catalytic subunit of a protein kinase called the M-phase promoting factor that induces entry into mitosis. CDK1 promotes G2-M transition, and regulates G1 progress and G1-S transition via association with multiple interphase cyclins (Cyclin A, Cyclin B). Dysregulated activation of CDK1, leading to uncontrolled cell division, is a hallmark of cancers. The inhibition of CDK1 is thus an active research area, especially since the emergence of new approaches involving PROTACs (PROteolysis TArgeting Chimeras). 

Benefits

  • SPECIFICITY
  • PRECISION
  • DATA NORMALIZATION

Total CDK1 assay principle

The Total CDK1 assay quantifies the expression level of CDK1 in a cell lysate. Unlike Western Blot, the assay is entirely plate-based and does not require gels, electrophoresis, or transfer. The Total CDK1 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 CDK1 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.

Principle of the HTRF total CDK1 assay

Total CDK1 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 CDK1 HTRF detection reagents. This protocol enables the cells' viability and confluence to be monitored.

2-plate protocol of the HTRF total CDK1 assay

Total CDK1 one-plate assay protocol

Detection of Total CDK1 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.
1-plate protocol of the HTRF total CDK1 assay

Validation of the specificity of Total CDK1 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 CDK1 detection antibodies. An additional 4 µL of lysates (supplemented with 12 µL diluent #11) was also transferred into the microplate to monitor the 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 CDK1 siRNA led to a 81% signal decrease compared to the cells transfected with the negative siRNA. No signal decrease was observed with cells transfected with CDK2, CDK4, CDK6, CDK7, CDK9, or CDK12 siRNAs. The  level of GAPDH measured remained unchanged under all the conditions tested. The data demonstrate that the HTRF Total CDK1 is specific for the detection of the CDK1 protein,and does not cross-react with other CDK family members.

siRNA experiments on Total CDK1 assay
siRNA experiments on Total CDK1 assay

Assessment of total CDK1 levels in human cell lines

Various human cell lines, either adherents HeLa,MCF7, SHSY5Y cells or suspension such as K562 cells, were seeded at 100,000 cells / well in a 96-well microplate. After 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 CDK1 detection reagents. The HTRF signal was recorded after an overnight incubation.

Total CDK1 assay validation on human cells

HTRF Total CDK1 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 CDK1 detection reagents. Equal amounts of lysates were used for a side by side comparison between HTRF and Western Blot.

Using the HTRF total CDK1 assay, 1250 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 CDK1 assay is 8 times more sensitive than  the Western Blot technique.

Comparison between HTRF and WB sensitivity on Total CDK1

Role of CDK1 in the cell-division cycle

CDK1 (Cyclin-Dependent Kinase 1) is a member of the subfamily of CDKs that coordinate cell cycle progression in mammalian cells (also including CDK2, CDK4, and CDK6).

Mitogenic signals, such as growth factors, trigger cells to enter the G1 phase of the cell cycle by inducing cyclin D synthesis, leading to the formation of active CDK4/6-cyclin D complexes. CDK4 and CDK6 mono-phosphorylate the protein of retinoblastoma (RB), which still binds to transcription factor E2F, but some genes can be transcribed, such as cyclin E. In the late G1 and early S phases, Cyclin E interacts with and activates CDK2, which in turn phosphorylates additional sites on RB, resulting in its complete inactivation. The E2F-responsive genes required for S phase progression are thus induced, such as Cyclin A which then interacts with CDK2 to form Cyclin A/CDK2 complexes. Activated CDK2 finally phosphorylates Cdc25B & Cdc25C phosphatases, which in turn activate CDK1, required for progression in the G2 and M phases of the cell-division cycle (centrosome maturation and separation, chromosome condensation and mitotic entry after nuclear envelope breakdown).

Total CDK1 pathway

HTRF cellular phospho-protein assays

Physiologically relevant results fo fast flowing research - Flyers

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Insider Tips for successful sample treatment - Technical Notes

Optimize your HTRF cell signaling assays on tissues

HTRF and WB compatible guidelines - Technical Notes

Best practices for analyzing tumor xenografts with HTRF phospho assays

Protocol for tumor xenograft analysis with HTRF - Technical Notes

Key guidelines to successful cell signaling experiments

Mastering the art of cell signaling assays optimization - Guides

HTRF® cell signaling platform combined with iCell® Hepatocytes

A solution for phospho-protein analysis in metabolic disorders - Posters

HTRF phospho-assays reveal subtle drug-induced effects

Detailed protocol and direct comparison with WB - Posters

Universal HTRF® phospho-protein platform: from 2D, 3D, primary cells to patient derived tumor cells

Analysis of a large panel of diverse biological samples and cellular models - Posters

HTRF phospho assays reveal subtle drug induced effects in tumor-xenografts

Tumor xenograft analysis: HTRF versus Western blot - Application Notes

HTRF cell-based phospho-protein data normalization

Valuable guidelines for efficiently analyzing and interpreting results - Application Notes

HTRF phospho-total lysis buffer: a universal alternative to RIPA lysis buffers

Increased flexibility of phospho-assays - Application Notes

HTRF Alpha-tubulin Housekeeping kit

Properly interpret your compound effect - Application Notes

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How to run a cell based phospho HTRF assay

What to expect at the bench - Videos

Unleash the potential of your phosphorylation research with HTRF

A fun video introducing you to phosphorylation assays with HTRF - Videos

How to run a cell based phospho HTRF assay

3' video to set up your Phospho assay - Videos

Guidelines for Cell Culture and Lysis in Different Formats Prior to HTRF Detection

Seeding and lysing recommendations for a number of cell culture vessels. - Technical Notes

Assessment of drug efficacy and toxicity by combining innovative technologies

Combination of AlphaLISA®, HTRF®, or AlphaLISA® SureFire® Ultra™ immunoassays with the ATPlite™ 1step cell viability assay - Application Notes

Product Insert Total CDK1 Kit / 64CDK1TPEG-64CDK1TPEH

64CDK1TPEG-64CDK1TPEH - Product Insert

Methodological Aspects of Homogeneous Time-Resolved Fluorescence (HTRF)

Learn how to reduce time and sample consumption - Application Notes

Plate Reader Requirement

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