Recombinant Human Active FAK Protein, CF

R&D Systems | Catalog # 4467-KS

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Key Product Details

  • R&D Systems Sf 9 (baculovirus)-derived Recombinant Human Active FAK Protein (4467-KS)
  • Quality control testing to verify active proteins with lot specific assays by in-house scientists
  • All R&D Systems proteins are covered with a 100% guarantee

Source

Sf 9 (baculovirus)

Accession Number

NM_153831

Applications

Bioactivity
View all FAK Proteins and Enzymes »

Product Specifications

Source

Spodoptera frugiperda, Sf 9 (baculovirus)-derived human FAK protein

Purity

>75%, by SDS-PAGE under reducing conditions and visualized by Colloidal Coomassie® Blue stain at 5 μg per lane.

N-terminal Sequence Analysis

Using an N-terminal His tag

SDS-PAGE

35 kDa

Activity

The specific activity of FAK was determined to be 51 nmol/min/mg as per Activity Assay Protocol, and was equivalent to 291.5 nmol/min/mg as per radiometric assay.

Scientific Data Images for Recombinant Human Active FAK Protein, CF

Recombinant Human Active FAK Protein SDS-PAGE

Recombinant Human Active FAK Protein SDS-PAGE.

The approximate molecular weight is 35 kDa and the purity is > 75%.

Formulation, Preparation, and Storage

4467-KS
Formulation Supplied in 50 mM Sodium Phosphate (pH 7.0), 300 mM NaCl, 150 mM Imidazole, 0.1 mM PMSF, 0.25 mM DTT, 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 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.

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). Elevated FAK expression in anaplastic astrocytoma and glioblastoma tumor biopsy samples has been demonstrated.

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

Alternate Names

FADK1, PTK2

Entrez Gene IDs

5747 (Human); 14083 (Mouse); 25614 (Rat)

Gene Symbol

PTK2

UniProt

NM_153831

Additional FAK Products

Product Documents for Recombinant Human Active FAK Protein, CF

Certificate of Analysis

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Note: Certificate of Analysis not available for kit components.

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Product Specific Notices for Recombinant Human Active FAK Protein, CF

For research use only

Citations for Recombinant Human Active FAK Protein, CF

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Protocols

View specific protocols for Recombinant Human Active FAK Protein, CF (4467-KS):

Materials
  • Active Kinase - Active FAK (0.05 μg/μL) diluted with Kinase Dilution Buffer X (1X) to the concentrations indicated in Figure 2. Note: These are suggested working dilutions. Optimal dilutions should be determined by each laboratory for each application.
  • Kinase Assay Buffer III (5X) - 200 mM Tris-HCl, pH 7.4, 100 mM MgCl2, and 0.5 mg/mL BSA. Add fresh DTT prior to use to a final concentration of 250 μM.
  • Kinase Dilution Buffer X (1X) - 12.5 mM MnCl2 diluted at a 1:4 ratio (5X dilution) with cold water. Add fresh DTT to the aliquot prior to use to a final concentration of 50 μM.
  • ADP-Glo™ Kinase Assay Kit - 10 mM ATP Solution, 10 mM ADP Solution, ADP-Glo™ Reagent, and Kinase Detection Reagent.
  • Substrate - Poly (Glu:Tyr, 4:1) synthetic peptide substrate was diluted in distilled water to a final concentration of 1 mg/mL.
  • Cofactor: 2.5 M MnCl2 - Diluted to a working concentration of 0.1 M in distilled water.
  1. Thaw the Active FAK, Kinase Assay Buffer III (5X), and Substrate on ice. Prepare a 15 μL enzyme dilution with Kinase Dilution Buffer X (1X) at the desired concentration, with Kinase Dilution Buffer X (1X), in a pre-chilled 96-well plate.
  2. Prepare a Substrate/ATP mixture as follows (25 μM example):
    a. 10 mM ATP Solution: 1.25 μL
    b. Kinase Assay Buffer III (5X): 46.75 μL
    c. Substrate at 1 mg/mL: 50 μL
    d. 0.1 M MnCl2: 2 μL
  3. Transfer the following reaction components prepared in Step 2 to a 384-well opaque plate, bringing the reaction volume up to 5 μL:
    a. 3 μL of diluted Active FAK
    b. 2 μL of Substrate/ATP mix as prepared in Step 2. This initiates the reaction.
  4. Set up the blank control as outlined in Step 2, excluding the addition of the kinase. Replace the kinase with an equal volume of Kinase Dilution Buffer X (1X).
  5. Incubate at ambient temperature for 40 minutes.
  6. After the 40 minute incubation period, terminate the reaction and deplete the remaining ATP by adding 5 μL of ADP-Glo™ Reagent. Spin down and shake the 384-well plate. Then incubate the reaction mixture for another 40 minutes at ambient temperature.
  7. Add 10 μL of the Kinase Detection Reagent to the 384-well plate and incubate the reaction mixture for another 30 minutes at ambient temperature.
  8. Read the 384-well reaction plate using the Luminescence Module Protocol on a GloMax®-Multi Microplate Multimode Reader.
  9. Determine the corrected activity (RLU) by removing the blank control value (see step 4) for each sample and calculate the kinase specific activity as outlined below.


    Calculation of Specific Activity of ADP (RLU/pmol)
    From ADP standard curve, determine RLU/pmol of ADP

    Kinase Specific Activity (SA) (pmol/min/μg or nmol/min/mg)
    Corrected RLU from reaction / [(SA of ADP in RLU/pmol) x (Reaction time in min) x (Enzyme amount in μg or mg)]

FAQs

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Associated Pathways

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