Not available outside of the UK & Ireland.

Analysis Note

ControlUntreated A-431 (negative control) and EGF treated A-431 (positive control) whole cell lysates.
Included Positive Antigen Control: Catalog # 12-302, EGF-stimulated A431 cell lysate is provided as a free positive antigen control for western immunoblotting. Aliquot as desired, refreeze immediately, and store at -20°C. The lysate is stable for 6 months at -20°C. Before use, add 2.5 µL of 2-mercaptoethanol/100 µL of lysate and boil for 5 minutes to reduce the preparation. Load 20 µg of reduced lysate per lane for immunoblot analysis.

Application

Research CategorySignaling

Research Sub CategoryGeneral Post-translation Modification

Anti-Phosphotyrosine Antibody, clone 4G10 detects tyrosine phosphorylated proteins in all species. This unique monoclonal antibody is validated for use in IC, IH, IP, WB and is backed by hundreds of publications

Immunoprecipitation:
2-4 µg of a previous lot immunoprecipitated quantitatively the phosphotyrosine containing proteins in the lysate of a confluent culture (10 cm dish) of cells expressing an activated tyrosine kinase. To preserve phosphotyrosine, add: 0.2 mM sodium orthovanadate to the lysis buffer.

Disclaimer

Unless otherwise stated in our catalog or other company documentation accompanying the product(s), our products are intended for research use only and are not to be used for any other purpose, which includes but is not limited to, unauthorized commercial uses, in vitro diagnostic uses, ex vivo or in vivo therapeutic uses or any type of consumption or application to humans or animals.

General description

Some of the tyrosine residues can be tagged with a phosphate group (phosphorylated) by protein kinases. (In its phosphorylated state, it is referred to as phosphotyrosine.). Tyrosine phosphorylation is considered as one of the key steps in signal transduction and regulation of enzymatic activity.
The advent of anti-phosphotyrosine antibodies is one of significant events in signal transduction research. Before the availability of anti-phosphotyrosine antibodies, tyrosyl phospyhorylation of proteins and enzymes was investigated through hazardous and time-consuming radioactive experiments. Anti-phosphotyrosine antibodies are commonly used in western blots after the targeted proteins have been immunoprecipitated to measure the tyrosyl phosphorylation of the proteins. Anti-phosphotyrosine antibodies are also directly used on cell lysate to examine the overall change of tyrosine phosphorylation level in reponse to various treatments.

Immunogen

Phosphotyramine-KLH

Legal Information

4G10 is a registered trademark of Upstate Group, Inc.

UPSTATE is a registered trademark of Merck KGaA, Darmstadt, Germany

Linkage

Replaces: MAB3090

Other Notes

Concentration: Please refer to the Certificate of Analysis for the lot-specific concentration.

Physical form

0.1M Tris-Glycine, 0.15M NaCl, 0.05% Sodium Azide, pH 7.4. Liquid at 2-8°C.
IgG2bκ mouse monocolonal antibody produced in vitro by mouse-mouse hybridoma 4G10™ (FOX-NY [NS-1 derivative] myeloma x spleen cells).

Protein G-Sepharose™ Chromatography

Format: Purified

Quality

Routinely evaluated on EGF-treated human A431 carcinoma cells.

Western Blot Analysis:
0.5-2 µg/mL of this lot detected tyrosine-phosphorylated proteins in a modified RIPA lysate from EGF-treated human A431 carcinoma cells (Cohen, B., 1990; , Druker, B. J., 1989; Kanakura, Y., 1991).

Specificity

This antibody recognizes tyrosine-phosphorylated proteins from all species.

Storage and Stability

Stable for 1 year at 2-8°C from date of receipt.

NOTE: DO NOT FREEZE.
For maximum recovery of the product, centrifuge the original vial prior to removing the cap. If the product has accidentally been frozen and thawed, spin it at 13,000 x g for 10 minutes at 2-8°C. Save the supernatant for application.

Target description

Dependent upon the molecular weight of the tyrosine phosphorylated protein being detected.