Antithrombin III/Serpin C1 Protein, Cynomolgus (HEK293, His)

Antithrombin III, also known as Serpin C1, forms a protease inhibitory heterodimer with TMPRSS7. This interaction exemplifies the regulatory role of antithrombin III as a serine protease inhibitor, highlighting its ability to modulate TMPRSS7 activity. Antithrombin III/Serpin C1 Protein, Cynomolgus (HEK293, His) is the recombinant cynomolgus-derived Antithrombin III/Serpin C1 protein, expressed by HEK293 , with C-His labeled tag.

Antithrombin III (ATIII), also known as Serpin C1, is a crucial serine protease inhibitor that forms a protease inhibiting heterodimer with TMPRSS7. This interaction underlines ATIII's role in regulating coagulation processes by inhibiting the activity of various proteases involved in the blood clotting cascade. The formation of a heterodimer with TMPRSS7 emphasizes the specificity of ATIII in targeting and modulating the activity of specific proteases. This protease inhibiting function plays a central role in preventing excessive blood clotting and maintaining hemostatic balance.

Measured by its ability to inhibit Recombinant Human Coagulation Factor II/Thrombin cleavage of a fluorogenic peptide substrate Boc-VPR-AMC. The IC₅₀ value is 2.185 nM, as measured under the described conditions.

Materials
Assay buffer: 50 mM Tris, 10 mM CaCl₂, 150 mM NaCl, 0.05% (w/v) Brij-35, pH 7.5 (TCNB)
Thrombin (MW 37kDa) (HY-114164)
Antithrombin III/Serpin C1 Protein, Cynomolgus (HEK293, His) (HY-P75581)
Heparin (HY-17567A): Diluted in deionized water to 20 mg/mL
Substrate: BOC-Val-Pro-Arg-AMC (HY-137784), dissolved in DMSO to 10 mM

Procedure
1. Dilute Thrombin to 1 μg/mL in assay buffer (with a final concentration of 48.6 μg/mL heparin) .
2. Prepare different concentrations of Antithrombin III in assay buffer with the following serial dilutions: 0, 2.31, 6.49, 13.9, 27.8, 41.7, 62.5, 125 nM.
3. Mix equal volumes of Antithrombin III (various concentrations) with Thrombin/Heparin (from step 1) . For the control group, add equal volumes of assay buffer and Thrombin/Heparin.
4. Incubate the mixture at room temperature for 30 minutes.
5. After incubation, dilute the mixture 1/5 with assay buffer.
6. Dilute the substrate to 200 μM in assay buffer.
7. Experimental Group: Mix 50 μL of the diluted mixture (from step 5) with 50 μL of 200 μM substrate.
Control Group: Mix 50 μL of assay buffer with 50 μL of 200 μM substrate.
8. Read the fluorescence in kinetic mode for 5 minutes with excitation and emission wavelengths of 380 nm and 460 nm (top read) .
9. Determine the 50% inhibitory concentration (IC₅₀) of Antithrombin III by plotting RFU/min versus concentration and fitting the data with a 4-parameter logistic (4-PL) curve.

Cynomolgus

HEK293

C-His

A0A2K5UYN7 (S35-S464)

102117641  [NCBI]

Full Length of Mature Protein

SPVDICTAKPRDIPMNPMCIYRSPEKKATEDEGSEQKIPEATNRRVWELSKANSRFATTFYQHLADSKNDKDNIFLSPLSVSTAFAMTKLGACNDTLKQLMEVFKFDTISEKTSDQIHFFFAKLNCRLYRKANKSSKLVSANRLFGDKSLTFNETYQDISELVYGAKLQPLDFKENAEQSRAAINKWVSNKTEGRITDVIPPEAINELTVLVLVNTIYFKGLWKSKFSPENTRMEPFYKADGESCSASMMYQEGKFRYRRVAEGTQVLELPFKGDDITMVLILPKPEKSLTKVEQELTPEVLQEWLDELEEMMLVVHMPRFRIEDGFSLKEQLQDMGLVDLFSPEKSKLPGIVAEGRDDLYVSDAFHKAFLEVNEEGSEAAASTAIGIAGRSLNPNRVTFKANRPFLVFIREVPLNTIIFMGRVANPCVS

Approximately 55-75 kDa

• 
  ≥ 95%, as determined by reducing SDS-PAGE.

Lyophilized powder.

Lyophilized from a 0.22 μm filtered solution of PBS, pH 7.4.

<1 EU/μg, determined by LAL method.

It is not recommended to reconstitute to a concentration less than 100 μg/mL in ddH₂O. For long term storage it is recommended to add a carrier protein (0.1% BSA, 5% HSA, 10% FBS or 5% Trehalose).

Stored at -20°C for 2 years from date of receipt. After reconstitution, it is stable at 4°C for 1 week or -20°C for longer (with carrier protein). It is recommended to freeze aliquots at -20°C or -80°C for extended storage.

Room temperature in continental US; may vary elsewhere.