Phosphotyrosine (0) antibody

Antigen: Phosphotyrosine (0)

Clonality: Monoclonal (13F9)

Host: Mouse

Application: ELISA, Western Blotting (WB), Immunoprecipitation (IP)

Catalog no.: ABIN105624

Additional Information

Alternative name: Phosphotyrosine (pY)

Immunogen: This monoclonal antibody was produced after repeated immunizations of balb/c mice with phosphotyrosine conjugated KLH. Hybridoma: Produced by the fusion between mouse splenocytes and mouse myeloma SP2/0 cells using conventional hybridoma technology.

Format: Liquid (sterile filtered)

Isotype: IgG1 kappa  (Matching secondary antibodies)

Clone: 13F9

Specificity: This product is clarified ascites produced in balb/c mice using clone 13F9. Reactivity is specific for phosphotyrosine and minimal cross reactivity is observed against phosphoserine or phosphothreonine.

Application Details

Application Notes: Recommended Dilutions: This product was assayed by against phosphotyrosine conjugated BSA by ELISA using HRP Goat-anti-Mouse IgG for detection. A working dilution of 1:2,000 to 1:10,000 is suggested for this assay. For immunoblotting dilute the antibody 1:1000 immediately before use. Researchers should determine optimal titers for other applications. Note: This monoclonal antibody reacts specifically with phosphotyrosine and shows minimal reactivity by ELISA and competitive ELISA with phosphoserine or phosphothreonine. The antibody reacts with free phosphotyrosine, phosphotyrosine conjugated to carriers such as thyroglobulin or BSA, and detects the presence of phosphotyrosine in proteins of both unstimulated and stimulated cell lysates. Although not tested, this antibody is likely functional in RIA, flow cytometry, immunohistochemistry and immunoprecipitation. Phosphorylation of tyrosine residues is associated with many growth factors and oncogene protein kinases, and is important for cell signaling in activation, proliferation and differentiation.

Buffer: Stabilizer: None. Preservative: 0.02% (w/v) Sodium Azide. Buffer: None.

Storage: Store vial at -20° C prior to opening. Aliquot contents and freeze at -20° C or below for extended storage. Avoid cycles of freezing and thawing. Centrifuge product if not completely clear after standing at room temperature. This product is stable for several weeks at 4° C as an undiluted liquid. Dilute only prior to immediate use. Expiration date is one (1) year from date of opening.

Research Area: Amino Acids, Protein Modifications

Restrictions: For Research Use only

Images

Mab anti-phosphotyrosine antibody (clone 13F9) is shown to detect by immunoblot (lane 1) phosphorylated EGFR in an A431 cell lysate (10 ug per lane) after stimulation with EGF. The binding of EGF to EGFR causes rapid activation of its intrinsic autophosphorylation of multiple tyrosine residues in the cytoplasmic domain of the protein. This propagates the ERK1/2 signal pathway which regulates cell growth, survival, proliferation and differentiation. Lane 2 shows total protein in the lysate after coommassie staining. Lane 3 (not shown) shows minimal reactivity of the antibody with a lysate from an unstimulated A431 cell lysate. A 4-20% gradient gel was used for separation prior to transfer to nitrocellulose. A 1:1,000 dilution of Mab antiphosphotyrosine is used at room temperature for 60 min followed by detection using IRDye

ELISA results of Mab antiphosphotyrosine antibody tested against BSA conjugates of pT, pY and pS. Each well was coated with 0.1ug of conjugate. The starting dilution of antibody was 1:1000 and each point on the Y-axis represents a 2-fold dilution. HRP conjugated Gt-a-Mouse IgG H&L and TMB substrate were used for detection.