Product Pathways - Motif Antibodies

Phospho-Tyrosine Mouse mAb (P-Tyr-100) (Alexa Fluor® 647 Conjugate) #9415

Applications	Reactivity	Source	Isotype
IF-IC F	All	Mouse	IgG1

Applications Key: IF-IC=Immunofluorescence (Immunocytochemistry) F=Flow Cytometry
Reactivity Key: All=All species expected

Specificity / Sensitivity

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-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 derived from mice immunized with phospho-tyrosine-containing peptides (KLH-coupled). The antibody was conjugated to Alexa Fluor® 647 under optimal conditions with an F/P ratio of 2-6.

Flow Cytometry

Flow cytometric analysis of K562 cells, untreated (green) or Gleevec®- treated (blue), using Phospho-Tyrosine Mouse mAb (P-Tyr-100) (Alexa Fluor® 647 Conjugate) compared with a nonspecific negative control antibody (red).

IF-IC

Confocal immunofluorescent image of two Gefinitib (Iressa) treated non-small cell lung cancer cell lines. HCC827 cells have the E746_A750 deletion in exon 19 of the EGFR gene, and are highly sensitive to Gefitinib. H1975 cells have the (T790M) mutation that confers Gefitinib-resistance. Both cell lines were treated and then double-labeled with Phospho-S6 Ribosomal Protein Rabbit mAb (Alexa Fluor® 488 Conjugate) #4854 and Phospho-Tyrosine Mouse mAb (Alexa Fluor® 647 Conjugate) #9415. Untreated HCC827 (A) and H1975 (C) cells show bright phospho-S6 (green) and phospho-tyrosine (red pseudocolor) label. Phospho-S6 and phospho-tyrosine signals dramatically decrease following Gefitinib treatment in HCC827 cells (B), with little or no change in the Gefitinib-resistant H1975 cells (D).

Description

Cell Signaling Technology Antibody conjugated to Alexa Fluor® 647 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® 647 conjugate) will also recognize phospho-tyrosine in all species.

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 Mouse mAbs developed by CST (P-Tyr-100, #9411 and P-Tyr-102, #9416) provide exceptionally sensitive new tools of increased utility for studying tyrosine phosphoyrlation and monitoring tyrosine kinase activity in high throughput drug discovery.

Application References

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Companion Products

9414 Phospho-Tyrosine Mouse mAb (P-Tyr-100) (Alexa Fluor® 488 Conjugate)
9411 Phospho-Tyrosine Mouse mAb (P-Tyr-100)

Alexa Fluor® is a registered trademark of Molecular Probes, Inc.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 commerical licensing terms please contact CST Business Development at cbunker@cellsignal.com.