Recombinant Human Active Lck Protein, CF Summary
CF stands for Carrier Free (CF). We typically add Bovine Serum Albumin (BSA) as a carrier protein to our recombinant proteins. Adding a carrier protein enhances protein stability, increases shelf-life, and allows the recombinant protein to be stored at a more dilute concentration. The carrier free version does not contain BSA.
In general, we advise purchasing the recombinant protein with BSA for use in cell or tissue culture, or as an ELISA standard. In contrast, the carrier free protein is recommended for applications, in which the presence of BSA could interfere.
|Formulation||Supplied in 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 10 mM glutathione, 0.25 mM DTT, 0.1 mM EDTA, 0.1 mM PMSF, 25% glycerol.
|Shipping||The product is shipped with dry ice or equivalent. Upon receipt, store it immediately at the temperature recommended below.|
|Stability & Storage:||This product is stable at ≤ ‑70 °C for up to 1 year from the date of receipt. For optimal storage, aliquot into smaller quantities after centrifugation and store at recommended temperature. Avoid repeated freeze-thaw cycles.|
- Active Kinase - Active Lck (0.1 μg/μL) diluted with Kinase Dilution Buffer and assayed as outlined in Figure 2. Note: These are suggested working dilutions. Optimal dilutions should be determined by each laboratory for each application.
- Kinase Assay Buffer I - 25 mM MOPS, pH 7.2, 12.5 mM beta -glycerolphosphate, 25 mM MgCl2, 5 mM EGTA, 2 mM EDTA. Add 0.25 mM DTT to the Kinase Assay Buffer prior to use.
- Kinase Dilution Buffer III - Kinase Assay Buffer I diluted at a 1:4 ratio (5-fold dilution) with 50 ng/μL BSA solution.
- 10 mM ATP Stock Solution - Prepare the ATP Stock Solution by dissolving 55 mg of ATP in 10 mL of Kinase Assay Buffer I. Store 200 μL aliquots at ≤ -20 °C.
- [33P]-ATP Assay Cocktail - Prepare 250 μM [33P]-ATP Assay Cocktail in a designated radioactive work area by combining 150 μL of 10 mM ATP Stock Solution, 100 μL of [33P]-ATP (1 mCi/100 μL), and 5.75 mL of Kinase Assay Buffer I. Store 1.0 mL aliquots at ≤ -20 °C.
- Substrate - Poly (Glu:Tyr, 4:1) synthetic peptide substrate diluted in distilled water to a final concentration of 1.0 mg/mL.
- Thaw the [33P]-ATP Assay Cocktail in a shielded container in a designated radioactive work area.
- Thaw the Active Lck, Kinase Assay Buffer I, Substrate, and Kinase Dilution Buffer on ice.
- In a pre-cooled microfuge tube, add the following reaction components bringing the initial reaction volume up to 20 μL.
a. Diluted Active Lck: 10 μL
b. Substrate (1.0 mg/mL Stock Solution): 5 μL
c. Distilled water: 5 μL
- Set up the blank control as outlined in step 3, excluding the addition of the substrate. Replace the substrate with an equal volume of distilled water.
- Initiate the reaction by the addition of 5 μL [33P]-ATP Assay Cocktail, bringing the final volume up to 25 μL. Incubate the mixture in a water bath at 30 °C for 15 minutes.
- After the 15 minute incubation period, terminate the reaction by spotting 20 μL of the reaction mixture onto individual pre-cut strips of phosphocellulose P81 paper.
- Air dry the pre-cut P81 strip and sequentially wash in a 1% phosphoric acid solution (add 10 mL of phosphoric acid to 990 mL of distilled or deionized water) with constant gentle stirring. It is recommended that the strips be washed a total of three times for approximately 10 minutes each.
- Count the radioactivity on the P81 paper in the presence of scintillation fluid in a scintillation counter.
- Determine the corrected cpm by removing the blank control value (see step 4) for each sample and calculate the kinase specific activity as outlined below:
Calculation of [33P]-ATP Specific Activity (SA) (cpm/pmol)
Specific Activity (SA) = cpm for 5 μL [33P]-ATP/pmole of ATP (in 5 μL of a 250 μM ATP stock solution; i.e. 1250 pmol)
Calculation of Kinase Specific Activity (SA) (pmol/minutes/μg or nmol/minutes/mg)
Corrected cpm from reaction / [(SA of 33P-ATP in cpm/pmol) x (Reaction time in minutes) x (Enzyme amount in μg or mg)] x [(Reaction volume) / (Spot Volume)]
The approximate molecular weight is 84 kDa and the purity is > 95%.
Lck (p56lck) is a member of the Src family of non-receptor tyrosine kinases. It was identified as a gene rearranged and over-expressed in the murine lymphoma LSTRA, most likely as a result of the insertion of Moloney murine leukemia virus DNA immediately adjacent to the gene (1). Lck behaves as a proto-oncogene and can lead to cell transformation upon activation. A number of human cancer cell lines show over-expression of Lck, pointing to a possible role for this kinase in the initiation and maintenance of the transformed state in human cancers (2).
- Fischer, S. et al. (1987) Biochem. Biophys. Res. Commun. 143:819.
- Veillette, A. et al. (1987) Oncogene Res. 1:357.
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