Flow cytometric analysis of Jurkat cells, untreated (blue) or treated with Anisomycin #2222 (25 ug/ml, 30 min; green), using Phospho-ATF-2 (Thr71)/ATF-7 (Thr53) (A8J7P) Rabbit mAb (PE Conjugate) (solid lines) or a concentration-matched Rabbit (DA1E) mAb IgG XP® Isotype Control (PE Conjugate) #5742 (dashed lines).
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This Cell Signaling Technology antibody is conjugated to phycoerythrin (PE) and tested in-house for direct flow cytometric analysis in human cells. This antibody is expected to exhibit the same species cross-reactivity as the unconjugated Phospho-ATF-2 (Thr71)/ATF-7 (Thr53) (A8J7P) Rabbit mAb #15411.
Supplied in PBS (pH 7.2), less than 0.1% sodium azide and 2 mg/ml BSA. Store at 4°C. Do not aliquot the antibody. Protect from light. Do not freeze.
All reagents required for this protocol may be efficiently purchased together in our Intracellular Flow Cytometry Kit (Methanol) #13593, or individually using the catalog numbers listed below.
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.
NOTE: When including fluorescent cellular dyes in your experiment (including viability dyes, DNA dyes, etc.), please refer to the dye product page for the recommended protocol. Visit www.cellsignal.com for a full listing of cellular dyes validated for use in flow cytometry.
NOTE: Adherent cells or tissue should be dissociated and in single-cell suspension prior to fixation.
NOTE: Optimal centrifugation conditions will vary depending upon cell type and reagent volume. Generally, 150-300g for 1-5 minutes will be sufficient to pellet the cells.
NOTE: If using whole blood, lyse red blood cells and wash by centrifugation prior to fixation.
NOTE: Antibodies targeting CD markers or other extracellular proteins may be added prior to fixation if the epitope is disrupted by formaldehyde and/or methanol. The antibodies will remain bound to the target of interest during the fixation and permeabilization process. However, note that some fluorophores (including PE and APC) are damaged by methanol and thus should not be added prior to permeabilization. Conduct a small-scale experiment if you are unsure.
NOTE: Count cells using a hemocytometer or alternative method.
posted July 2009
revised June 2020
Protocol Id: 407
Phospho-ATF-2 (Thr71)/ATF-7 (Thr53) (A8J7P) Rabbit mAb (PE Conjugate) detects endogenous levels of ATF-2 and ATF-7 only when phosphorylated at threonine 71 and threonine 53, respectively. This antibody does not cross-react with phosphorylated c-Jun, CREB, or other transcription factors. It recognizes Thr69/Thr71 dually phosphorylated ATF-2, Thr51/Thr53 dually phosphorylated ATF-7, Thr71 singly phosphorylated ATF-2, and Thr53 singly phosphorylated ATF-7 equally well.
Human, Mouse, Rat, Monkey
Monoclonal antibody is produced by immunizing animals with a synthetic phosphopeptide corresponding to residues surrounding Thr71 of human ATF-2 protein.
The transcription factor ATF-2 (also called CRE-BP1) binds to both AP-1 and CRE DNA response elements and is a member of the ATF/CREB family of leucine zipper proteins (1). ATF-2 interacts with a variety of viral oncoproteins and cellular tumor suppressors and is a target of the SAPK/JNK and p38 MAP kinase signaling pathways (2-4). Various forms of cellular stress, including genotoxic agents, inflammatory cytokines, and UV irradiation, stimulate the transcriptional activity of ATF-2. Cellular stress activates ATF-2 by phosphorylation of Thr69 and Thr71 (2-4). Both SAPK and p38 MAPK have been shown to phosphorylate ATF-2 at these sites in vitro and in cells transfected with ATF-2. Mutations of these sites result in the loss of stress-induced transcription by ATF-2 (2-4). In addition, mutations at these sites reduce the ability of E1A and Rb to stimulate gene expression via ATF-2 (2).
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