Cellomics HCS Reagent Kits for multiplex analysis of p53 and p21 expression.

Figure 1. Staining of p21 and p53 in A549 cells treated with media only (Untreated) or with 5 μM Camtothecin for 20 hrs. Proteins were labeled according to the kit protocol. The cells were imaged using a Cellomics ArrayScan HCS Reader.

Description

p53 is a tumor suppressor protein that regulates the expression of a wide variety of genes involved in apoptosis, growth arrest, inhibition of cell cycle progression, differentiation and accelerated DNA repair or senescence in response to genotoxic or cellular stress. p53 is maintained in normal cells at low levels. However, when the cell is confronted with stress like DNA damage, hypoxia, cytokines, metabolic changes, viral infection, or oncogenes, p53 accumulates in the nucleus, where it is activated. Once activated, p53 can function as a sequence-specific transcription factor that can act both directly and indirectly to turn on or off various genes that affect cell growth and survival. Additionally, p53 can directly interact with several proteins that influence growth and survival.

The cell cycle is regulated by several cyclins. These cyclins ensure successful transitions form S phase to G1. p53 is an upstream inducer of p21 which is an inhibitor of CDKs (Cyclin Dependent Kinase). Inhibition of these kinases results in the disruption of G1-to-S and G2-to-mitosis transition in cells. Mutations in p53 have been associated with genomic instability and increase susceptibility to cancer.

The Multiplexed p53 and p21 Detection HCS Reagent Kits are for the simultaneous quantitation of p53 and p21 expression in the same cell (Figure 1). These kits allow direct measurements of p53 and p21 in the nucleus using a fixed end-point assay based on immunofluorescence detection in cells grown on standard high-density microplates. The primary antibodies are specific for their targets and have minimal cross-reactivity with other targets. Prepared cells can be analyzed using standard fluorescence microscopy or using Cellomics ArrayScan HCS Reader with the Molecular Translocation BioApplication, affording automated plate handling, focusing, image acquisition, analysis, quantification, and data storage.

Features

• Available in multiplex or singleplex configurations
• Optimized protocol requiring no cell lysis, purification or filtration steps
• Immunofluorescence-based assay provides sensitive results without radioactivity
• Cells prepared with the HCS Reagent Kits may be analyzed directly via standard fluorescence microscopy; or HCS Imaging Systems including the ArrayScan HCS Reader
• Primary antibodies are specific for their targets and have minimal cross-reactivity with other targets

Related Poster Presentations

	Ann F. Hoffman, Shannon M. Hamilton, Krishna M. Vatten, Janaki Narahari, and Richik N. Ghosh (2007) Evaluation of p53 Induction Using High Content Screening.
	Krishna M. Vattem, Janaki Narahari, Richik N. Ghosh (2007) New High Content Screening Reagent Kits to Monitor Induction of p53 and its Downstream Targets.
	Krishna M. Vattem, Janaki Narahari, Richik N. Ghosh (2006) High Content Analysis of Induction of p53 and its Downstream Targets.
	Sarah Burroughs Tencza and Jeffrey R. Haskins (2004) High-Content Profiling of Cell Cycle Related Targets with RNAi.
	Yih-Tai Chen, Kenneth A. Giuliano, and Jeffrey R. Haskins (2003) Validation of siRNA Gene Knockdown Through Automated Phenotypic Analysis of Cellular Function.

Kit Contents

• p53 primary antibody
• p21 primary antibody
• DyLight™ 549 conjugated goat anti-rabbit IgG
• DyLight™ 549 conjugated goat anti-mouse IgG
• Alexa Fluor^® 488 conjugated goat anti-mouse IgG
• Hoechst dye
• Wash buffer (10X Dulbecco's PBS)
• Permeabilization buffer (10X Dulbecco's PBS with 1% Triton^® X-100)
• Blocking buffer (10X)
• Thin plate seal

Ordering Information

Screening size kits and components (bulk and custom) available with special pricing. Please call.

To order Cellomics HCS Reagent Kits, please call 800-874-3723 in the U.S.
or contact your local distributor of Thermo Scientific Products.

Choose "Order Online" in table to see pricing and order online (U.S. only).

See all Cellomics HCS Reagent Kits