Universal Tyrosine Kinase Assay Kit
Applications:
- Quantitative Measurement of Tyrosine Kinase Activity
- Analysis of PTK Activity Regulation
- In vitro Screening of PTK Inhibitors
Description:
Protein Tyrosine Kinase (PTK) is an important enzyme of the signal transduction pathway which controls cell proliferation, differentiation and carcinogenesis. PTK is classified into two groups: 1) membrane receptor PTKs (e.g. insulin receptor and EGF receptor) and 2) non-receptor linked PTKs (e.g. Src and FAK). Receptor PTKs contain three domains: an extracellular domain, a membrane intrinsic domain, and an intracellular domain. The extracellular and membrane intrinsic domains bind to ligands such as hormones and growth factors, while the intracellular domain carries the PTK active site. Receptor PTKs transduce signals directly into the cell by phosphorylation of tyrosine when ligands bind to their domains. Non-receptor linked PTKs lack the membrane intrinsic domain and are divided into three types based upon their location in the cell (i.e. membranes, cytoplasm, or nucleus). Non-receptor linked PTKs participate in various functions, including transduction and cell-cell contact responses.
The Universal Tyrosine Kinase Assay Kit is an in vitro enzyme immunoassay kit for quantitative determination of tyrosine kinase activity using either adherent or suspension cell culture extracts, and is useful for analyzing regulation of PTK activity and for in vitro screening of PTK inhibitors. The kit utilizes a PTK substrate which is coated onto a 96-well microtiter plate, a phosphotyrosine (PY20)-horse radish eroxidase (HRP) antibody, and HRP substrate (TMBZ). Serial dilutions of a prepared cell extract are added with an ATP solution to the PTK substrate-coated microtiter plate, allowing phosphorylation of tyrosine. The sample solution is then removed, and the plate well washed and blocked. Following blocking, anti-phosphotyrosine (PY20)-HRP is added, incubated, and then replaced by the HRP substrate solution (TMBZ). Color development ensues, and the amount of sample PTK activity is determined colorimetrically by comparing the sample's specific absorbance at 450 nm with the absorbance obtained for the Standard plotted on a standard curve.
Kit Components:
| PTK substrate-immobilized microplate | 1 plate (8 wells x 12 strips) |
| Kinase reacting solution | 1 vial (1 x 11 mL) |
| 40 mM ATP-2Na (lyophilized) | 2 vials (for 2 x 0.55 mL) |
| Extraction buffer | 1 vial (1 x 11 mL) |
| PTK control (lyophilized) | 1 vial (for 1 x 0.5 mL) |
| Anti-phosphotyrosine (PY20)-HRP (lyophilized) | 1 vial (for 1 x 5.5 mL) |
| Blocking solution | 1 vial (1 x 11 mL) |
| HRP substrate solution (TMBZ) | 1 vial (1 x 12 mL) |
Performance Characteristics
| Range of assay: | 2.16-135 x 10-5 U/μL |
| Assay sensitivity: | 2.16 x 10-5 U/μL sample (= 32 fmol/well) (as Src kinase) |
| Assay duration: | 1.5-3 hours |
| Specificity: | γ-phosphate residue transfer from ATP to substrate. No reactivity with phosphorylated serine or threonine. |
| Test specimen type: | Suspension or adherent cell extracts. |
| Sample volume per well: | 40 μL |
Storage:
2–8°C
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