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.
Product Information
Format
Purified
Control
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.
Presentation
Purified mouse monoclonal IgG2bκ in buffer containing PBS, pH 7.4. Liquid at 2-8oC. IgG2bκ mouse monoclonal antibody produced in vitro by mouse-mouse hybridoma 4G10? (FOX-NY [NS-1 derivative] myeloma x spleen cells).
Storage and Shipping Information
Storage Conditions
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.
Applications
Application
Anti-Phosphotyrosine Antibody, clone 4G10 detects level of Phosphotyrosine & has been published & validated for use in IP & WB.
Key Applications
Immunoprecipitation
Western Blotting
Application Notes
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.
Biological Information
Immunogen
Phosphotyramine-KLH
Clone
4G10?
Concentration
Please refer to the Certificate of Analysis for the lot-specific concentration.
Host
Mouse
Specificity
This antibody recognizes tyrosine-phosphorylated proteins from all species.
Isotype
IgG2bκ
Species Reactivity
All
Antibody Type
Monoclonal Antibody
Modifications
Phosphorylation
Purification Method
Protein G-Sepharose? Chromatography
Molecular Weight
Dependent upon the molecular weight of the tyrosine phosphorylated protein being detected.
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