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HTRF Human p21 Detection kit HTRF®

The HTRF Human p21 Detection kit is designed to detect the expression level of p21 in cell lysates.

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  • All inclusive kit All inclusive kit
  • No-wash No-wash
  • Low sample consumption Low sample consumption

The HTRF Human p21 Detection kit is designed to detect the expression level of p21 in cell lysates.

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Overview

p21, also known as cyclin-dependent kinase inhibitor 1 or CDK-interacting protein 1, is a key cyclin-CDK complex inhibitor that binds to cyclin-CK2, cyclin-CDK1, and cyclin-CDK4/6, and regulates their activity. p21 works in tandem with p53 to achieve downstream cell-cycle arrest following DNA damage, and is negatively regulated by ubiquitin-ligase-driven protein degradation.

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HTRF Human p21 assay principle

The HTRF Human p21 assay quantifies the expression level of human p21 in a cell lysate. Unlike Western Blot, the assay is entirely plate-based and does not require gels, electrophoresis, or transfer. The assay uses two labeled antibodies: one coupled to a donor fluorophore, the other to an acceptor. In presence of p21 in a cell lysate, 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 Human p21 assay

HTRF Human p21 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 human p21 HTRF detection reagents. This protocol enables the cells' viability and confluence to be monitored.

2-plate protocol of the HTRF Human p21 assay

p21 activation in Tenovin-1-stimulated MCF-7

MCF-7 cells were seeded at 50,000 cell per well under 100 µl in a 96-well plates in complete culture medium and incubated overnight at 37°C, 5% CO2. After incubation, cells were treated with a dose-response of the p53 activator Tenovin-1, and incubated for 24h at 37°C, 5% CO2.

After incubation, culture medium was removed and cells were lysed with 50 µL of supplemented lysis buffer #2 at 1X for 30 minutes at RT under gentle shaking.

16 µL of lysate were transferred into a low volume white microplate before the addition of 2 µL of the HTRF d2 detection reagent and 2 µL HTRF Eu-K detection reagent. The HTRF signal was recorded after a 4h incubation.

Validation of the HTRF Human p21 assay in MCF-7 cells

p21 simplified pathway

p21 mediates cell-cycle arrest in tandem with p53 (p53-dependant signaling). This p53-p21 axis is activated in response to multiple signals related to cell health and DNA damage. Some p21 promoters include pro-inflammatory pathway products, like JNK or p38, but also stress from radical oxygen species (ROS). The regulation of pro-apoptosis signaling, cell-cycle arrest, and DNA repair roles that p21 and p53 fulfill promote strong anti-proliferative processes and  make them master effectors of several tumor suppressor pathways.

Simplified pathway of p21

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

Methodological Aspects of Homogeneous Time-Resolved Fluorescence (HTRF)

Learn how to reduce time and sample consumption - Application Notes

Product Insert P21 Kit / 64HP21PEG-64HP21PEH

64HP21PEG-64HP21PEH - Product Insert

Plate Reader Requirement

Choosing the right microplate reader ensures you’ll get an optimal readout. Discover our high performance reader, or verify if your lab equipment is going to be compatible with this detection technology.

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