Phosphothreonine (pT) antibody

Antigen: Phosphothreonine (pT)

Clonality: Monoclonal (18F6)

Host: Mouse

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

Catalog no.: ABIN105601

Additional Information

Immunogen: This monoclonal antibody was produced after repeated immunizations of balb/c mice with phosphothreonine 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: 18F6

Specificity: This product is clarified ascites produced in balb/c mice using clone 18F6. Reactivity is specific for phosphothreonine and minimal cross reactivity is observed against phosphoserine or phosphotyrosine.

Application Details

Application Notes: Recommended Dilutions: This product was assayed by against phosphothreonine 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 phosphothreonine and shows minimal reactivity by ELISA and competitive ELISA with phosphoserine or phosphotyrosine. The antibody reacts with free phospho amino acid, phosphothreonine conjugated to carriers such as thyroglobulin or BSA, and detects the presence of phosphothreonine 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 threonine 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: 0.01% (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

ELISA results of Mab antiphosphothreonine 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.

Mab anti-phosphothreonine antibody (clone 18F6) is shown to detect threonine phosphorylation of proteins in a lysate after EGF stimulation of A431 cells. Separation is achieved under reducing conditions using a pre-cast 4-20% iGel from Gradipore Inc. A 1:1,000 dilution of Mab anti-phosphothreonine is used for 30 min followed by detection using a 1:2,500 dilution of IRDyeTM800 conjugated Goat-a-Mouse IgG [H&L] (610- 132-121) and visualization using the Odyssey