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3939
Phospho-Stat5 (Tyr694) (C71E5) Rabbit mAb (Alexa Fluor® 488 Conjugate)

Phospho-Stat5 (Tyr694) (C71E5) Rabbit mAb (Alexa Fluor® 488 Conjugate) #3939

APPLICATIONS

REACTIVITY SENSITIVITY MW (kDa) Isotype
H M Endogenous Rabbit IgG
Flow Cytometry

Flow cytometric analysis of TF-1 cells, untreated (blue) or treated with GM-CSF (green), using Phospho-Stat5 (Tyr694) (C71E5) Rabbit mAb (Alexa Fluor® 488 Conjugate).

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Flow Cytometry, Methanol Permeabilization Protocol for Direct Conjugates

A. Solutions and Reagents

NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.

  1. 20X Phosphate Buffered Saline (PBS): (#9808) To prepare 1 L 1X PBS: add 50 ml 20X PBS to 950 ml dH2O, mix.
  2. 16% Formaldehyde (methanol free).
  3. 100% methanol.
  4. Incubation Buffer: Dissolve 0.5 g Bovine Serum Albumin (BSA) (#9998) in 100 ml 1X PBS. Store at 4°C.

B. Fixation

NOTE: If using whole blood, lyse red blood cells and wash by centrifugation prior to fixation.

  1. Collect cells by centrifugation and aspirate supernatant.
  2. Resuspend cells in 0.5-1 ml 1X PBS. Add formaldehyde to obtain a final concentration of 4%.
  3. Fix for 15 min at room temperature.
  4. Wash by centrifugation with excess 1X PBS. Discard supernatant in appropriate waste container. Resuspend cells in 0.5-1 ml 1X PBS.

C. Permeabilization

  1. Permeabilize cells by adding ice-cold 100% methanol slowly to pre-chilled cells, while gently vortexing, to a final concentration of 90% methanol.
  2. Incubate 30 min on ice.
  3. Proceed with immunostaining (Section D) or store cells at -20°C in 90% methanol.

D. Immunostaining

  1. Aliquot desired number of cells into tubes or wells.
  2. Wash cells by centrifugation in excess 1X PBS to remove methanol. Discard supernatant in appropriate waste container. Repeat if necessary.
  3. Resuspend cells in 100 µl of diluted antibody conjugate (prepared in incubation buffer at the recommended dilution).
  4. Incubate for 1 hr at room temperature. Protect from light.
  5. Wash by centrifugation in incubation buffer. Discard supernatant. Repeat.
  6. Resuspend cells in 1X PBS and analyze on flow cytometer; alternatively, for DNA staining, proceed to optional DNA stain (Section E).

E. Optional DNA Dye

  1. Resuspend cells in 0.5 ml of DNA dye (e.g. Propidium Iodide (PI)/RNase Staining Solution #4087).
  2. Incubate for at least 5 min at room temperature.
  3. Analyze cells in DNA staining solution on flow cytometer.

posted July 2009

revised June 2017

Protocol Id: 407

Application Dilutions
Flow Cytometry 1:50
Storage:

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.

Phospho-Stat5 (Tyr694) (C71E5) Rabbit mAb (Alexa Fluor® 488) detects endogenous levels of Stat5a only when phosphorylated at Tyr694 and Stat5b when phosphorylated at Tyr699.

Species Reactivity:

Human, Mouse

Species predicted to react based on 100% sequence homology:

Rat, Monkey, Bovine

Monoclonal antibody is produced by immunizing animals with a synthetic phosphopeptide corresponding to residues surrounding Tyr694 of Stat5a. The antibody was conjugated to Alexa Fluor® 488 under optimal conditions with an F/P ratio of 2-6.

This Cell Signaling Technology (CST) antibody is conjugated to Alexa Fluor® 488 fluorescent dye and tested in-house for direct flow cytometry in human cells. CST expects that Phospho-Stat5 (Tyr694) (C71E5) Rabbit mAb (Alexa Fluor® 488) will display the same species cross-reactivity as the unconjugated antibody (Phospho-Stat5 (Tyr694) (C71E5) Rabbit mAb #9314).

Stat5 is activated in response to a wide variety of ligands including IL-2, GM-CSF, growth hormone and prolactin. Phosphorylation at Tyr694 is obligatory for Stat5 activation (1,2). This phosphorylation is mediated by Src upon erythropoietin stimulation (3). Stat5 is constitutively active in some leukemic cell types (4). Phosphorylated Stat5 is found in some endothelial cells treated with IL-3, which suggests its involvement in angiogenesis and cell motility (5). Stat5a and Stat5b are independently regulated and activated in various cell types. For instance, interferon treatment predominantly activates Stat5a in U-937 cells and Stat5b in HeLa cells (6).

  1. Gouilleux, F. et al. (1994) EMBO J 13, 4361-9.
  2. Wakao, H. et al. (1994) EMBO J 13, 2182-91.
  3. Okutani, Y. et al. (2001) Oncogene 20, 6643-50.
  4. Demoulin, J.B. et al. (1999) J Biol Chem 274, 25855-61.
  5. Dentelli, P. et al. (1999) J. Immunol. 163, 2151-2159.
  6. Meinke, A. et al. (1996) Mol Cell Biol 16, 6937-44.
Entrez-Gene Id
6776 , 6777
Swiss-Prot Acc.
P42229 , P51692
For Research Use Only. Not For Use In Diagnostic Procedures.

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.
U.S. Patent No. 7,429,487, foreign equivalents, and child patents deriving therefrom.

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