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9414
Phospho-Tyrosine Mouse mAb (P-Tyr-100) (Alexa Fluor® 488 Conjugate)

Phospho-Tyrosine Mouse mAb (P-Tyr-100) (Alexa Fluor® 488 Conjugate) #9414

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APPLICATIONS

REACTIVITY SENSITIVITY MW (kDa) Isotype
H M R All Endogenous Mouse IgG1
Flow Cytometry
Flow Cytometry

Flow cytometric analysis of K562 cells, untreated (green) or Gleevec®- treated (blue), using Phospho-Tyrosine Mouse mAb (P-Tyr-100) (Alexa Fluor® 488 Conjugate) compared with a nonspecific negative control antibody (red).

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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-Tyrosine Mouse mAb (P-Tyr-100) is a high affinity antibody. ELISAs against a wide variety of phosphopeptides indicate that (a) P-Tyr-100 binds phospho-Tyr in a manner largely independent of the surrounding amino acid sequence. 2D gel Western blot analysis of pervanadate-treated cell extracts also shows that P-Tyr-100 interacts with a broad range of tyrosine-phosphorylated proteins. P-Tyr-100 does not cross-react with peptides containing phospho-serine or phospho-threonine. (U.S. Patent No's.: 6,441,140; 6,982,318; 7,259,022; 7,344,714; U.S.S.N. 11,484,485; and all foreign equivalents.)

Species Reactivity:

Human, Mouse, Rat, All Species Expected

Monoclonal antibody is produced by immunizing animals with phospho-tyrosine-containing peptides . The antibody was conjugated to Alexa Fluor® 488 under optimal conditions with an F/P ratio of 2-6.

Cell Signaling Technology Antibody conjugated to Alexa Fluor® 488 fluorescent dye and tested in-house for direct flow cytometric analysis of human cells.

*The unconjugated antibody, #9411, reacts with phospho-tyrosine in all species tested. CST expects that Phospho-Tyrosine Mouse mAb (P-Tyr-100) (Alexa Fluor® 488 conjugate) will also recognize phospho-tyrosine in all species.

Tyrosine phosphorylation plays a key role in cellular signaling (1). Research studies have shown that in cancer, unregulated tyrosine kinase activity can drive malignancy and tumor formation by generating inappropriate proliferation and survival signals (2). Antibodies specific for phospho-tyrosine (3,4) have been invaluable reagents in these studies. The phospho-tyrosine monoclonal antibodies developed by Cell Signaling Technology are exceptionally sensitive tools for studying tyrosine phosphorylation and monitoring tyrosine kinase activity in high throughput drug discovery.

  1. Schlessinger, J. (2000) Cell 103, 211-25.
  2. Blume-Jensen, P. and Hunter, T. (2001) Nature 411, 355-65.
  3. Ward, S.G. et al. (1992) J Biol Chem 267, 23862-9.
  4. Glenney, J.R. et al. (1988) J Immunol Methods 109, 277-85.
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.
Use of Cell Signaling Technology (CST) Motif Antibodies within certain methods (e.g., U.S. Patents No. 7,198,896 and 7,300,753) may require a license from CST. For information regarding academic licensing terms please have your technology transfer office contact CST Legal Department at CST_ip@cellsignal.com. For information regarding commercial licensing terms please contact CST Pharma Services Department at ptmscan@cellsignal.com.

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9414S		 100 µl (50 tests) $ 297.0

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