Cellomics HCS Reagent Kit for detection of C/EBP homologous protein (CHOP), also called GADD153.

control	treat cells	detect CHOP/ GADD153	analyze	conclude

Description

The Cellomics CHOP/GADD153 Detection Kit is for direct quantitation of CHOP/GADD153 expression in the nucleus (Figure 1). This kit uses a fixed end-point assay based on immunofluorescence detection in cells grown on standard high-density microplates and contains a primary antibody toward CHOP/GADD153 (mouse monoclonal) and a goat anti-mouse secondary antibody conjugated with DyLight 549 Fluorophore. The primary antibody is specific for its target and has minimal cross-reactivity with other targets.

Expression of mutant proteins disrupts protein folding in the endoplasmic reticulum (ER), causes ER stress and activates a signaling network called the unfolded protein response (UPR). Depending on the capacity of the ER to repair the protein folding process, the UPR either increases or decreases the biosynthetic capacity of the secretory pathway through upregulation of ER chaperone expression or by attenuating the global protein synthesis and increasing pro-apoptotic mechanisms. The C/EBP homologous protein (CHOP, also known as GADD153) is a transcription factor activated during ER stress by two pathways: the ATF4 pathway and the ATF6 pathway. CHOP-induced apoptotic mechanisms are mediated in part by CHOP-dependent decreased Bcl2 expression and increased transcriptional induction of the BH3-only protein Bim. Deregulated CHOP activity compromises cell viability.

CHOP/GADD153 expression in A549 cells treated with ER stress-inducing drugs was quantitatively assayed using the Cellomics CHOP/GADD153 Detection Kit, Cellomics ArrayScan HCS Reader (Figure 1) and the Cellomics Compartmental Analysis BioApplication Software Module. Thus, automated plate-handling, focusing, cell image acquisition/processing and data analysis/management are combined in one high-content screening (HCS) system to assay for test compounds regulating CHOP/GADD153 expression (Figure 2). In addition to HCS instruments, cells labeled by the reagents in this kit can be viewed and analyzed by fluorescence microscopes.

	Hoechst	CHOP/GADD153
Tunicamycin			Figure 1. Nuclear expression of CHOP/GADD153 increases significantly in cells treated with tunicamycin. Staining of CHOP/ GADD153 in A549 cells treated with media only (non-treated) or with 1.5 µM tunicamycin for 6 hours. Cells were stained according to the kit protocol with Hoechst Dye, CHOP/GADD153 Primary Antibody and DyLight 549 Conjugated Goat Anti-Mouse IgG. Stained cells were imaged using a Cellomics ArrayScan HCS Reader. Color thumnail images at top of page link to color-merged images of Hoechst (blue) and CHOP/GADD153 (orange) channels in non-treated (control) and treated cells.
Non-treated

Figure 2. Dose response curves of CHOP/GADD153 in A549 cells. The difference in average CHOP/GADD153 intensities of the nucleus and cytoplasm are plotted in response to cells treated with various drug concentrations for 6 hours. Tm: tunicamycin, EC50 = 1 µM. MG132 (proteasome inhibitor): EC50 = 1.3 µM. NaAr: sodium arsenite, EC50 = 32.2 µM. COX-2 inhibitor: N-(5-acetyl-2-piperidinophenyl)-N´-(2,5-dichlorophenyl)thiourea, EC50 = 32.2 µM.

Related Poster Presentations

Bhaskar S. Mandavilli, Krishna Vattem, and Richik N. Ghosh (2008) Automated, Quantitative, Multi-Parametric Monitoring of Different Programmed Cell Death Pathways Using Cell-Based High-Content Screening Assays.

Kit Contents

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).

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