Tyrosine Kinases / Adaptors

Phospho-EGF Receptor Pathway Antibody Sampler Kit #9789

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Item #	Description	List Price	Web Price	QTY	Buy
9789S	Phospho-EGF Receptor Pathway Antibody Sampler Kit - 1Kit	$645.00	$580.50

Protocols

9789: Western Blotting, Flow, IHC / Paraffin, Immunofluorescence, Western Blotting, Flow, IHC / Paraffin, Immunofluorescence, Immunoprecipitation, Western Blotting, Flow, IHC / Paraffin, Immunofluorescence, Immunoprecipitation, IHC / Frozen, Western Blotting, Flow, Immunoprecipitation, Western Blotting, Western Blotting, Western Blotting, Immunoprecipitation, Western Blotting, Flow, Immunofluorescence, Immunoprecipitation,

Specificity / Sensitivity

Each antibody in the Phospho-EGF Receptor Pathway Sampler Kit recognizes the phosphorylated form of its specific target. Phospho-EGF Receptor (Tyr1068) (D7A5) XP™ Rabbit mAb may cross-react weakly with other tyrosine-phosphorylated proteins. Phospho-Gab1 (Tyr627) (C32H2) Rabbit mAb may cross-react with phosphorylated Gab2, Gab3, or activated receptor tyrosine kinases. Phospho-Shc (Tyr239/240) Antibody may cross-react with activated EGF receptor protein.

Source / Purification

Activation state polyclonal antibodies are produced by immunizing animals with synthetic phosphopeptides corresponding to residues surrounding Tyr774 of human c-Cbl, Tyr239/240 of human Shc and Tyr783 of human PLCγ1. Polyclonal antibodies are purified by protein A and peptide affinity chromatography. Rabbit monoclonal antibodies are produced by immunizing animals with synthetic phosphopeptides corresponding to residues surrounding Tyr1068 of human EGF receptor, Tyr694 of Stat5a, Tyr627 of human Gab1, Ser473 of human Akt and Thr202/Tyr204 of human p44 MAP kinase.

Description

The Phospho-EGF Receptor Pathway Sampler Kit provides an economical means to evaluate the activation status of multiple members of the EGF receptor pathway, including phosphorylated EGF receptor, Stat5, c-Cbl, Shc, Gab1, PLCγ1, Akt and p44/42 MAPK. The kit includes enough primary and secondary antibodies to perform four western blot experiments.

Western Blotting

Western blot analysis of extracts of BxPC-3 cells, untreated or EGF-stimulated, using Phospho-EGF Receptor (Tyr1068) (D7A5) XP™ Rabbit mAb #3777 (upper) and EGF Receptor Antibody #2232 (lower).

Western Blotting

Western blot analysis of extracts from UT-7 cells, untreated or treated with erythropoietin (EPO; 3 units/ml for 5 min), TF-1 cells, untreated or treated with Human Granulocyte Macrophage Colony Stimulating Factor #8922 (hGM-CSF; 100 ng/ml for 10 min), and NK-92 cells, untreated or treated with Human Interleukin-2 #8907 (hIL-2; 100 ng/ml for 10 min), using Phospho-Stat5 (Tyr694) (D47E7) XP™ Rabbit mAb #4322 (upper) or total Stat5 (3H7) Rabbit mAb #9358 (lower).

Western Blotting

Western blot analysis of extracts from 293T cells transfected with c-Cbl and Fyn as indicated, untreated or calf intestinal phosphatase (CIP)-treated, using Phospho-c-Cbl (Tyr774) Antibody #3555. (Lysates provided by Dr. A.L. Reddi, laboratory of Dr. Hamid Band, Harvard University, Massachusetts.)

Western Blotting

Western blot analysis of extracts from Hep G2 cells, untreated or EGF-treated (100 ng/ml), and Jurkat cells, untreated or treated with anti-CD3 antibody (1 µg/ml for 10 minutes), using Phospho-Shc (Tyr239/240) Antibody #2434 (upper) or Shc Antibody #2432 (lower).

Western Blotting

Western blot analysis of cell extracts from T-47D cells, untreated or stimulated with heregulin, using Phospho-Gab1 (Tyr627) (C32H2) Rabbit mAb #3233 (upper) or Gab1 Antibody #3232 (lower).

Western Blotting

Western blot analysis of extracts from NIH/3T3 cells, untreated or PDGF-stimulated for the indicated times, using Phospho-PLCγ1 (Tyr783) Antibody #2821 (upper) or PLCγ1 Antibody #2822 (lower).

Western Blotting

Western blot analysis of extracts from PC-3 cells, untreated or LY294002/wortmannin-treated, and NIH/3T3 cells, serum-starved or PDGF-treated, using Phospho-Akt (Ser473) (D9E) XP™ Rabbit mAb #4060 (upper) or Akt (pan) (C67E7) Rabbit mAb #4691 (lower).

Western Blotting

Western blot analysis of extracts from 293, NIH/3T3 and C6 cells, treated with λ phosphatase or TPA as indicated, using Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (D13.14.4E) XP™ Rabbit mAb #4370 (upper), or p44/42 MAPK (Erk1/2) (137F5) Rabbit mAb #4695 (lower).

Background

The epidermal growth factor (EGF) receptor is a 170 kDa transmembrane tyrosine kinase that belongs to the HER/ErbB protein family. Ligand binding results in receptor dimerization, autophosphorylation, activation of downstream signaling, internalization and lysosomal degradation (1,2). Phosphorylation of EGF receptor (EGFR) at Tyr845 in the kinase domain is implicated in stabilizing the activation loop, maintaining the active state enzyme and providing a binding surface for substrate proteins (3,4). c-Src is involved in phosphorylation of EGFR at Tyr845 (5). The SH2 domain of PLCγ binds at phospho-Tyr992, resulting in activation of PLCγ-mediated downstream signaling (6). Phosphorylation of EGFR at Tyr1045 creates a major docking site for c-Cbl, an adaptor protein that leads to receptor ubiquitination and degradation following EGFR activation (7,8). The GRB2 adaptor protein binds activated EGFR at phospho-Tyr1068 (9). A pair of phosphorylated EGFR residues (Tyr1148 and Tyr1173) provides a docking site for the Shc scaffold protein, with both sites involved in MAP kinase signaling activation (2). Phosphorylation of EGFR at specific serine and threonine residues attenuates EGFR kinase activity. EGFR carboxy-terminal residues Ser1046 and Ser1047 are phosphorylated by CaM kinase II; mutation of either of these serines results in upregulated EGFR tyrosine autophosphorylation (10).

Application References

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This product is for in vitro research use only and is not intended for use in humans or animals. This product is not intended for use as therapeutic or in diagnostic procedures.