>90%, by SDS-PAGE under reducing conditions and visualized by silver stain.
<1.0 EU per 1 μg of the protein by the LAL method.
110 kDa. Disclaimer note: The observed molecular weight of the protein may vary from the listed predicted molecular weight due to post translational modifications, post translation cleavages, relative charges, and other experimental factors.
Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent
Activate rhEnterokinase with Thermolysin.
Dilute rhEnterokinase to 100 μg/mL in Assay Buffer.
Dilute Thermolysin to 3.16 μg/mL in Assay buffer.
Mix equal volumes of diluted rhEnterokinase and Thermolysin.
Incubate at 37 °C for 30 minutes.
Stop the reaction by adding an equal volume of 20 mM 1,10 Phenanthroline to the reaction tube.
Dilute rhEnterokinase to 0.1 μg/mL in Assay Buffer.
Dilute Substrate to 200 μM in Assay Buffer with 200 μM of DTNB.
Load in a 96 well clear plate 50 μL of the diluted rhEnterokinase. For a Substrate Blank load 50 μL of the Assay Buffer.
Start the reaction by adding 50 μL of the Substrate/DTNB mixture to wells.
Read in kinetic mode for 5 minutes at an absorbance of 405 nm.
Calculate specific activity:
Specific Activity (pmol/min/µg) =
Adjusted Vmax* (OD/min) x well volume (L) x 1012 pmol/M
ext. coeff** (M-1cm-1) x path corr.*** (cm) x amount of enzyme (µg)
*Adjusted for Substrate Blank
**Using the extinction coefficient 13260 M-1cm-1
***Using the path correction 0.32 cm
Note: the output of many spectrophotometers is in mOD
rhEnterokinase: 0.005 μg
DTNB: 100 μM
Substrate: 100 μM
This product is produced by and ships from R&D Systems, Inc., a Bio-Techne brand.
Alternate Names for Recombinant Human Enteropeptidase/Enterokinase Protein, CF
protease, serine, 7 (enterokinase)
Serine protease 7
Transmembrane protease serine 15
transmembrane protease, serine 15
EK initiates activation of pancreatic proteases by converting trypsinogen to trypsin, which in turn activates chymotrypsin, carboxypeptidases and elastases. Located in intestinal brush border, it is a disulfide bond linked dimer of the heavy and light chains, which are derived from the same single-chain precursor. The multidomain‑containing the heavy chain consists of a short cytoplamic tail, a transmembrane, a SEA, a SRCR, a MAM, two CUB and two LDL-receptor class A domains. The light chain contains the catalytic domain of trypsin-like serine proteases. The purified recombinant human EK corresponds to the single-chain form starting at the end of the transmembrane domain.
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