Flow cytometric analysis of ACHN cells, untreated (blue) or treated with hIL-4 (10 ng/mL, 10 min; green), using Phospho-Stat6 (Tyr641) (D8S9Y) Rabbit mAb (Alexa Fluor® 647 Conjugate) (solid lines) or concentration-matched Rabbit (DA1E) mAb IgG XP® Isotype Control (Alexa Fluor® 647 Conjugate) #2985 (dashed line).
This Cell Signaling Technology antibody is conjugated to Alexa Fluor® 647 fluorescent dye and tested in-house for direct flow cytometry in human cells. The antibody is expected to exhibit the same species cross-reactivity as the unconjugated Phospho-Stat6 (Tyr641) (D8S9Y) Rabbit mAb #56554.
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
Phospho-Stat6 (Tyr641) (D8S9Y) Rabbit mAb (Alexa Fluor® 647 Conjugate) recognizes endogenous levels of Stat6 protein only when phosphorylated at Tyr641. Weak cross-reactivity with other tyrosine-phosphorylated proteins has been observed in some cell lines.Species Reactivity:
Human, MouseSpecies predicted to react based on 100% sequence homology:
Monoclonal antibody is produced by immunizing animals with a synthetic phospho-peptide corresponding to residues surrounding Tyr641 of human Stat6 protein.
Upon activation by Janus kinases, Stat6 translocates to the nucleus where it regulates cytokine-induced gene expression. Stat6 is activated via phosphorylation at Tyr641 and is required for responsiveness to IL-4 and IL-13 (1-4). In addition, Stat6 is activated by IFN-α in B cells, where it forms transcriptionally active complexes with Stat2 and p48 (5,6). Protein phosphatase 2A is also involved in regulation of IL-4-mediated Stat6 signaling (7).
Explore pathways + proteins related to this product.
Cell Signaling Technology is a 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).