Recombinant Human Active FAK Protein, CF Summary
Accession # NM_153831
Using an N-terminal His tag
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 sodium phosphate (pH 7.0), 300 mM NaCl, 0.25 mM DTT, 150 mM imidazole, 0.1 mM PMSF, and 25% glycerol.
|Shipping||The product is shipped with dry ice or equivalent. Upon receipt, store it immediately at the temperature recommended below.|
|Stability & Storage:||Thisproduct is stable at ≤ ‑70° C for upto 1 year from the date of receipt. For optimal storage, aliquot into smallerquantities after centrifugation and store at recommended temperature. Avoid repeated freeze-thaw cycles.|
- Active Kinase - Active FAK (0.1 μg/μL) diluted with Kinase Dilution Buffer. Note: These are suggested working dilutions. Optimal dilutions should be determined by each laboratory for each application.
- Kinase Assay Buffer II, pH 7.2 - 25 mM MOPS, 12.5 mM beta -glycerolphosphate, 20 mM MgCl2, 12.5 mM MnCl2, 5 mM EGTA, 2 mM EDTA. Add 0.25 mM DTT to the Kinase Assay Buffer prior to use.
- Kinase Dilution Buffer, pH 7.2 - Kinase Assay Buffer II diluted 5-fold with a 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 II. 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 II. Store 1 mL aliquots at
≤ -20 °C.
- Substrate - Poly (Glu:Tyr; 4:1) synthetic peptide was diluted in distilled or deionized water to a final concentration of 1 mg/mL.
- Thaw the [33P]-ATP Assay Cocktail in a shielded container in a designated radioactive work area.
- Thaw the Active FAK, Kinase Assay Buffer II, 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 FAK: 10 μL
b. Poly Substrate (1 mg/mL; on ice): 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 or deionized water.
- Initiate the reaction with 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, 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 subtracting 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 35 kDa and the average purity is 80%.
FAK (Focal Adhesion Kinase) is a non-receptor protein tyrosine kinase involved in signal transduction from integrin-enriched focal adhesion sites that mediate cell contact with the extracellular matrix. FAK-enhanced signals have been shown to mediate the survival of anchorage-dependent cells and are critical for efficient cell migration in response to growth factor receptor and integrin stimulation (1). Elevated expression of FAK in human tumors has been correlated with increased malignancy and invasiveness (2).
- Schaller, M.D. (2001) Biochim. Biophys. Acta 1540:1.
- Gabarra-Niecko, V. et al. (2003) Cancer Metastasis Rev. 22:359.
Citation for Recombinant Human Active FAK Protein, CF
R&D Systems personnel manually curate a database that contains references using R&D Systems products. The data collected includes not only links to publications in PubMed, but also provides information about sample types, species, and experimental conditions.
1 Citation: Showing 1 - 1
Molecular Regulation of the RhoGAP GRAF3 and Its Capacity to Limit Blood Pressure In Vivo
Authors: RA Dee, X Bai, CP Mack, JM Taylor
Sample Types: Recombinant Protein
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