Product Pathways - Motif Antibodies

Phospho-Tyrosine Mouse mAb (P-Tyr-100) #9411

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Applications	Reactivity	Sensitivity	Isotype
W IP IHC-P IF-F IF-IC IF-P F E-P	All	Endogenous	Mouse IgG1

Applications Key: W=Western Blotting IP=Immunoprecipitation IHC-P=Immunohistochemistry (Paraffin) IF-F=Immunofluorescence (Frozen) IF-IC=Immunofluorescence (Immunocytochemistry) IF-P=Immunofluorescence (Paraffin) F=Flow Cytometry E-P=ELISA (Peptide)
Reactivity Key: All=All species expected

Protocols

9411:: ELISA Peptide, Flow, IHC / Paraffin, Immunofluorescence, Immunoprecipitation, Western Blotting

Specificity / Sensitivity

Phospho-Tyrosine Mouse mAb (P-Tyr-100) is a high affinity antibody. ELISAs against a wide variety of phosphopeptides indicate that 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-Ser or phospho-Thr. (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.)

Source / Purification

Monoclonal antibody is produced by immunizing animals with phospho-tyrosine containing peptides .

Western Blotting

Western blot analysis of extracts from Jurkat cells treated with 1 mM pervanadate for 30 minutes prior to lysis, using P-Tyr-100 Phospho-Tyrosine Mouse mAb. Proteins were separated by 2-D electrophoresis prior to blotting.

IHC-P (paraffin)

Immunohistochemical analysis of paraffin-embedded human breast carcinoma using Phospho-Tyrosine Mouse mAb (P-Tyr-100).

IHC-P (paraffin)

Immunohistochemical analysis of paraffin-embedded human lung carcinoma using Phospho-Tyrosine Mouse mAb (P-Tyr-100).

IHC-P (paraffin)

Immunohistochemical analysis of paraffin-embedded NCI-H1650 xenograft untreated (left) or lambda-phosphatase-treated (right), using Phospho-Tyrosine Mouse mAb (P-Tyr-100).

Flow Cytometry

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

IF-IC

Immunofluorescent analysis of Swiss NIH/3T3 cells, serum-starved and stimulated with lysophosphatidic acid (LPA) (10 µM for 10 minutes) and fixed with PFA, using Phospho-Tyrosine Mouse mAb (P-Tyr-100) (red) and phalloidin for F-actin (green) LPA causes heavy tyrosine phosphorylation of proteins in focal adhesions, present at the tips of actin stress fibers. (Provided by Dr. Harry Mellor, University of Bristol.)

IF-IC

Confocal immunofluorescence analysis of HeLa cells either stimulated with 20% serum (left) or untreated (right), using Phospho-Tyrosine Mouse mAb (P-Tyr-100) (red). Actin filaments have been labeled with fluorescein phalloidin. Blue pseudocolor = DRAQ5™ (fluorescent DNA dye).

IF-F

Confocal immunofluorescent analysis of paraffin-embedded human lung adenocarcinoma using Phospho-Tyrosine Mouse mAb (P-Tyr-100) (red). Blue pseudocolor = DRAQ5™ (fluorescent DNA dye).

ELISA-Peptide

Phospho-Tyrosine Mouse mAb (P-Tyr-100) ELISA Assay: Signal-to-noise ratio of phospho- versus nonphospho-peptides. (Y* denotes phosphorylated tyrosine.)

Background

Tyrosine phosphorylation plays a key role in cellular signaling (1). 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 provide exceptionally sensitive new tools of increased utility for studying tyrosine phosphorylation and monitoring tyrosine kinase activity in high throughput drug discovery.

Application References

Smith, J.A. et al. (2005) Cancer Res 65, 1027-34. Applications: Western Blotting
Ye, Z.J. et al. (2005) Proc Natl Acad Sci U S A 102, 18461-6. Applications: Western Blotting
Sharma, A. et al. (2003) Mol Cell Proteomics 2, 1217-24. Applications: IP Western Blotting
Li, Y. et al. (2003) Mol Cell Biol 23, 6255-66. Applications: IP Western Blotting
Kogata, N. et al. (2003) Mol Biol Cell 14, 3553-64. Applications: Western Blotting
del Rincón, S.V. et al. (2003) Oncogene 22, 3353-60. Applications: Western Blotting
Goldberg, G.S. et al. (2003) J Biol Chem 278, 46533-40. Applications: Western Blotting
Klein, R.D. and Fischer, S.M. (2002) Carcinogenesis 23, 217-21. Applications: Western Blotting
Perez, O.D. and Nolan, G.P. (2002) Nat Biotechnol 20, 155-62. Applications: Flow Cytometry
Kaluz, S. et al. (2002) Cancer Res 62, 4469-77. Applications: IP
Kato, R. et al. (2002) Clin Cancer Res 8, 2455-62. Applications: IP
Asao, H. et al. (2001) J Immunol 167, 1-5. Applications: Western Blot
Stewart, D.M. et al. (2001) Endocrinology 142, 98-107. Applications: Western Blotting
Farhang-Fallah, J. et al. (2000) J Biol Chem 275, 40492-7. Applications: Western Blot
Chang, C.I. et al. (2000) J Immunol 165, 2134-41. Applications: Western Blot

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License/Use Restrictions: Use of CST Motif Antibodies within certain methods (e.g., U.S. Patent No.'s 7,198,896 & 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 Business Development at cbunker@cellsignal.com.

This product is intended for research purposes only. The product is not intended to be used for therapeutic or diagnostic purposes in humans or animals.