Recombinant Human Active FAK Protein, CF

Catalog # Availability Size / Price Qty
4467-KS-010
Recombinant Human Active FAK Protein SDS-PAGE
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Recombinant Human Active FAK Protein, CF Summary

Product Specifications

Purity
>70%, by SDS-PAGE under reducing conditions and visualized by Colloidal Coomassie® Blue stain at 5 μg per lane
Activity
The activity of FAK is typically 221-299 nmol/min/mg using a poly (Glu:Tyr, 4:1) synthetic peptide substrate (see Activity Assay Protocol).
Source
Spodoptera frugiperda, Sf 9 (baculovirus)-derived human FAK protein
Accession # NM_153831
Accession #
N-terminal Sequence
Analysis

Using an N-terminal His tag

SDS-PAGE
35 kDa

Product Datasheets

Carrier Free

What does CF mean?

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.

What formulation is right for me?

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.

4467-KS

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.

Assay Procedure

Materials
  • 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.
  1. Thaw the [33P]-ATP Assay Cocktail in a shielded container in a designated radioactive work area.
  2. Thaw the Active FAK, Kinase Assay Buffer II, Substrate, and Kinase Dilution Buffer on ice.
  3. 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
  4. 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.
  5. 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.
  6. 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.
  7. 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.
  8. Count the radioactivity on the P81 paper in the presence of scintillation fluid in a scintillation counter.
  9. 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)]

Data Image

SDS-PAGE Recombinant Human Active FAK Protein SDS-PAGE View Larger

The approximate molecular weight is 35 kDa and the average purity is 80%.

Reconstitution Calculator

Reconstitution Calculator

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Background: FAK

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).

References
  1. Schaller, M.D. (2001) Biochim. Biophys. Acta 1540:1.
  2. Gabarra-Niecko, V. et al. (2003) Cancer Metastasis Rev. 22:359.
Long Name
Focal adhesion kinase 1
Entrez Gene IDs
5747 (Human); 14083 (Mouse); 25614 (Rat)
Alternate Names
EC 2.7.10; FADK 1; FADK1; FADKFAK-related non-kinase polypeptide; FAK; FAK1FRNK; FAKEC 2.7.10.2; focal adhesion kinase 1; pp125FAK; Protein-tyrosine kinase 2; PTK2 protein tyrosine kinase 2; PTK2

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

  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
    Cells, 2020;9(4):.
    Species: N/A
    Sample Types: Recombinant Protein
    Applications: Bioassay

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