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.
This antibody recognizes tyrosine-phosphorylated proteins from all species.
Research Sub Category
General 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
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.
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).
Dependent upon the molecular weight of the tyrosine phosphorylated protein being detected.
Protein G-Sepharose Chromatography
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).
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 2-8ºC. Save the supernatant for application.
Untreated 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.
Concentration: Please refer to the Certificate of Analysis for the lot-specific concentration.
4G10 is a registered trademark of Upstate Group, Inc.
UPSTATE is a registered trademark of Merck KGaA, Darmstadt, Germany
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.