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.
Source
Spodoptera frugiperda, Sf 9 (baculovirus)-derived human FAK protein
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.
Buffer
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.
Purity
>75%, by SDS-PAGE under reducing conditions and visualized by Colloidal Coomassie® Blue stain at 5 μg per lane.
Assay Procedure
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.
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.
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
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.
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).
Incubate at ambient temperature for 40 minutes.
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.
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.
Read the
384-well reaction plate using the Luminescence Module Protocol on a
GloMax®-Multi Microplate Multimode Reader.
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)]
Notes
This product is produced by and ships from R&D Systems, Inc., a Bio-Techne brand.
Alternate Names for Recombinant Human Active FAK Protein, CF
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
Background
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.
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