Flow cytometric analysis of LNCaP cells (blue) and Jurkat cells (green) using NFAT1 (D43B1) XP® Rabbit mAb (Alexa Fluor® 488 Conjugate) (solid lines) or a concentration-matched Rabbit (DA1E) mAb IgG XP® Isotype Control (Alexa Fluor® 488 Conjugate) #2975 (dashed lines).
This Cell Signaling Technology antibody is conjugated to Alexa Fluor® 488 fluorescent dye and tested in-house for direct flow cytometry analysis in human cells. The antibody is expected to exhibit the same species cross-reactivity as the unconjugated NFAT1 (D43B1) XP® Rabbit mAb #5861.
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/flowdyes for a 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 August 2019
Protocol Id: 407
NFAT1 (D43B1) XP® Rabbit mAb (Alexa Fluor® 488 Conjugate) detects endogenous levels of total NFAT1 protein.Species Reactivity:
Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Gly87 of human NFAT1 protein.
The NFAT (nuclear factor of activated T cells) family of proteins consists of NFAT1 (NFATc2 or NFATp), NFAT2 (NFATc1 or NFATc), NFAT3 (NFATc4), and NFAT4 (NFATc3 or NFATx). All members of this family are transcription factors with a Rel homology domain and regulate gene transcription in concert with AP-1 (Jun/Fos) to orchestrate an effective immune response (1,2). NFAT proteins are predominantly expressed in cells of the immune system, but are also expressed in skeletal muscle, keratinocytes, and adipocytes, regulating cell differentiation programs in these cells (3). In resting cells, NFAT proteins are heavily phosphorylated and localized in the cytoplasm. Increased intracellular calcium concentrations activate the calcium/calmodulin-dependent serine phosphatase calcineurin, which dephosphorylates NFAT proteins, resulting in their subsequent translocation to the nucleus (2). Termination of NFAT signaling occurs upon declining calcium concentrations and phosphorylation of NFAT by kinases such as GSK-3 or CK1 (3,4). Cyclosporin A and FK506 are immunosuppressive drugs that inhibit calcineurin and thus retain NFAT proteins in the cytoplasm (5).
Explore pathways + proteins related to this product.
Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
XP is a registered trademark of Cell Signaling Technology, Inc.
Alexa Fluor is a registered trademark of Life Technologies Corporation.
The Alexa Fluor dye conjugates in this product are sold under license from Life Technologies Corporation, for research use only excluding use in combination with DNA microarrays and high content screening (HCS).
This product is provided under an intellectual property license from Life Technologies Corporation. The transfer of this product is conditioned on the buyer using the purchased product solely in research conducted by the buyer, excluding contract research or any fee for service research, and the buyer must not (1) use this product or its components for (a) diagnostic, therapeutic or prophylactic purposes; (b) testing, analysis or screening services, or information in return for compensation on a per-test basis; or (c) manufacturing or quality assurance or quality control, and/or (2) sell or transfer this product or its components for resale, whether or not resold for use in research. For information on purchasing a license to this product for purposes other than as described above, contact Life Technologies Corporation, 5791 Van Allen Way, Carlsbad, CA 92008 USA or firstname.lastname@example.org.