Flow cytometric analysis of THP1 cells, untreated (blue) or anisomycin-treated (green), using Phospho-SAPK/JNK (T183/Y185) (G9) Mouse mAb (Alexa Fluor®647 Conjugate) (#9257) compared to a nonspecific control antibody (red).
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Cell Signaling Technology Antibody conjugated to Alexa Fluor® 647 fluorescent dye and tested in-house for direct Flow Cytometric analysis of human and mouse cells. The unconjugated antibody #9255 reacts with, among others, human, mouse, rat and hamster phospho-SAPK/JNK (Thr183/Tyr185). CST expects that Phospho-SAPK/JNK (Thr183/Tyr185) (G9) Mouse mAb (Alexa Fluor® 647 Conjugate) will also recognize phospho-SAPK/JNK (Thr183/Tyr185) in these species.
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-SAPK/JNK (Thr183/Tyr185) (G9) mAb (Alexa Fluor® 647 Conjugate) detects endogenous levels of p46 and p54 SAPK/JNK dually phosphorylated at Thr183 and Tyr185. This antibody does not recognize endogenous levels of phosphorylated p44/42 MAPK or p38 MAP kinase.Species Reactivity:
Human, Mouse, Rat, Hamster, S. cerevisiae
Monoclonal antibody is produced by immunizing animals with a synthetic phosphopeptide corresponding to residues around Thr183/Tyr185 of human SAPK/JNK. The antibody was conjugated to Alexa Fluor® 647 under optimum conditions with an F/P ratio of 2-6. This antibody was conjugated to Alexa Fluor® 647 under optimal conditions with an F/P ratio of 2-6. The Alexa Fluor® 647 dye is maximally excited by red light (e.g. 633 nm He-Ne laser). Antibody conjugates of the Alexa Fluor® 647 dye produce bright far-red-fluorescence emission, with a peak at 665 nm.
The stress-activated protein kinase/Jun-amino-terminal kinase SAPK/JNK is potently and preferentially activated by a variety of environmental stresses including UV and gamma radiation, ceramides, inflammatory cytokines, and in some instances, growth factors and GPCR agonists (1-6). As with the other MAPKs, the core signaling unit is composed of a MAPKKK, typically MEKK1-MEKK4, or by one of the mixed lineage kinases (MLKs), which phosphorylate and activate MKK4/7. Upon activation, MKKs phosphorylate and activate the SAPK/JNK kinase (2). Stress signals are delivered to this cascade by small GTPases of the Rho family (Rac, Rho, cdc42) (3). Both Rac1 and cdc42 mediate the stimulation of MEKKs and MLKs (3). Alternatively, MKK4/7 can be activated in a GTPase-independent mechanism via stimulation of a germinal center kinase (GCK) family member (4). There are three SAPK/JNK genes each of which undergoes alternative splicing, resulting in numerous isoforms (3). SAPK/JNK, when active as a dimer, can translocate to the nucleus and regulate transcription through its effects on c-Jun, ATF-2, and other transcription factors (3,5).
Explore pathways + proteins related to this product.
Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
The Alexa Fluor dye antibody conjugates in this product are sold under license from Life Technologies Corporation for research use only, except for use in combination with DNA microarrays. The Alexa Fluor® dyes (except for Alexa Fluor® 430 dye) are covered by pending and issued patents. Alexa Fluor® is a registered trademark of Molecular Probes, Inc.