T9247-25 is a direct ELISA system for the colorimetric detection of protein tyrosine phosphatase activity from a variety of sources including crude preparations such as cell lysates or column fractions, immune complexes containing the tyrosine kinase of interest, or pure recombinant enzyme. By virtue of the strong biotin-streptavidin interaction (Kd ~10e-15 M), the peptide substrate is efficiently captured, and the stringency of washes can be adjusted as needed to help reduce non-specific background in ELISA.
This non-radioactive kinase assay kit is a two-step peptide phosphorylation/colorimetric detection assay kit. In the first stage, a biotinylated substrate peptide containing tandem repeats of Poly (Glu4-Tyr) is incubated with a tyrosine kinase enzyme sample in the presence of non-radioactive ATP and a Mn2+/Mg2+ co-factor cocktail. The kinase assay can be performed with substrate peptide either in solution, or bound to the well of a streptavidin-coated 96-well plate. The second step involves the detection of phosphorylated substrate by Direct Enzyme Linked Immunosorbent Assay (ELISA) using a monoclonal anti-phosphotyrosine-HRP (Horse Radish Peroxidase) antibody conjugate. Tetramethylbenzidine (TMB) is provided as the HRP substrate. Also provided with this kt is a phosphorylated Poly (Glu4-Tyr) biotinylated peptide which can be added to a reference well, thereby serving as a positive control for the direct ELISA portion of this assay.
To assay the tyrosine kinase activity of enzymes immobilized on a solid support (e.g., kinase samples in immune complexes boudn to agarose beads) the in vitro kinase assay must be performed with the peptide in solution, followed by peptide binding to the plate. An alternative procedure is included in this manual. Proten A-agarose or Protein G-agarose required for capturing immune complexes are user-supplied. Additional Tyrosine Kinase Reaction Buffer, 5X, and the Ply (Glu4-Tyr) Peptide, biotin conjugate can be purchased since the immune-complex protocol requires larger amounts of these components.
The user should determine the linear range of the assay empirically. This assay can be performed at room temperature (25 degrees C), 30 degrees C, or 37 degrees C, with incubation times up to 30 minutes. Further incubation or higher levels of incorporation may not be linear and may therefore not give a true representation of kinase activity.
Althouh tyrosine kinases do not exhibit tight specificity for particular substrates, the Poly(Glu4-Tyr) substrate peptide included in the kit is phosphorylated by many tyrosine kinases, some may not recognize the peptide as a substrate. Each kit contains sufficient reagents for 96 individual assays.
A recombinant kinase (active Src) has been used for internal testng of this assay kit.
T9247-25A: Streptavidin Coated Microwell Plate, 1x12 strips of 8 wells each
T9247-25B: Poly (Glu4-Tyr) Peptide (Biotin), 1 x12.5ug One vial containing 12.5ug peptide in 250ul sterile distilled water. Description: Fifty-four residue synthetic peptide [GG(EEEEY)10 EE] containing biotin at the N-terminus.
Molecular Weight = 7413 Daltons Purity: >90% determined by reverse-phase HPLC.
T9247-25C: Poly (Glu4-Phospho-Tyr) Peptide (Biotin), 1x5ug. One vial containing 5ug in 50ul sterile 0.1M sodium bicarbonate. Description: Twenty-seven residue synthetic phospho-peptide [GG(EE[pY]EE)5] containing a biotin at the N-terminus. Molecular Weight =4155 Daltons Purity: Greater than 90% determined by reverse-phase HPLC.
T9247-25D: Tyrosine Kinase Reaction Buffer (5X), 1x5ml. 5X Tyrosine Kinase Reaction Buffer (TKRB): 100mM Tris-HCl, pH 7.4, 50mM MgCl2, 5mM MnCl2, 5mM dithiothreitol, 1mM ATP.
T9247-25E: Sodium Orthovanadate, 50mM, 1x500ul. Supplied as a liquid in 50mM Sodium Orthovanadate, pH 10.0.
T9247-25F: TBS, 2% Tween?-20, 20X , 1x50ml. Supplied as a liquid in 1M Tris, 3M sodium chloride, 2% Tween- 20, pH 7.4.
T9247-25G: 10% BSA in TBS, 1x10ml. Supplied as a liquid in 10% BSA in 50mM Tris 150mM NaCl, pH 7.4
T9247-25H: Anti-phosphotyrosine, recombinant 4G10TM, (HRP), 1x25ul. Supplied as a liquid in containing 25ul of IgG2bk- HRP conjugate in PBS, 5mg/ml BSA, 0.05% Kathon?. Description: Recombinant 4G10TM, produced in Chinese Hamster Ovary (CHO) cells expressing the 4G10TM antibody heavy and light chain cDNAs. Heavy chain C-terminus has a hexa-histidine (6His) tag for purification and immobilization via nickel affinity matrices. Cross-linked to horseradish peroxidase (HRP). Patent pending. Immunogen: Phosphotyramine conjugated to KLH.
T9247-25I: TMB Substrate Reagent A, 1x7.5ml. Supplied as a liquid in 0.04% (w/v) Tetramethylbenzidine solution.
T9247-25J: TMB Substrate Reagent B: 1x7.5ml. Supplied as a liquid in 0.02% hydrogen peroxide in citric acid buffer.
Storage and Stability:
Upon receipt, store individual components at recommended temperatures. Store components T9247-25B, T9247-25C, T9247-25D and T9247-25E at -20 degrees C. Store all other components at 4 degrees C. Components are stable for 6 months from date of shipment if stored and handled correctly.