Serpin A4 蛋白, Human (HEK293, His)

Serpin A4 Protein inhibits tissue kallikrein's amidolytic and kininogenase activities by forming a heat- and SDS-stable equimolar complex with the enzyme. Tissue kallikrein concurrently cleaves the reactive site of Serpin A4, producing a small C-terminal fragment. The protein, existing as a monomer and occasionally forming homodimers, demonstrates its regulatory role in modulating tissue kallikrein-mediated enzymatic activities. Serpin A4 Protein, Human (HEK293, His) is the recombinant human-derived Serpin A4 protein, expressed by HEK293 , with C-6*His labeled tag.

Serpin A4 蛋白作为抑制剂，专门针对组织激肽释放酶的酰胺分解和激肽原酶活性。这种抑制机制涉及抑制剂和酶之间形成等摩尔复合物，该复合物对热和 SDS 稳定。同时，组织激肽释放酶裂解丝氨酸蛋白酶抑制剂 A4 的反应位点，产生一个小的 C 末端片段。该蛋白质以单体形式存在，在某些情况下形成同二聚体，显示出其在调节组织激肽释放酶介导的酶活性中的调节作用。

Measured by its ability to inhibit KLK1( HY-P73262) cleavage of a colorimetric peptide substrate, Pro-Phe-Arg-7-amido-4-methylcoumarin(HY-P2618) that incubate at 37°C for 60 minutes. The IC₅₀ value is ≤7.54 nM. (Activation description: The proenzyme needs to be activated by Thermolysin for an activated form.)

Materials
Activation buffer: 50 mM Tris, 10 mM CaCl₂, 150 mM NaCl, pH 7.5 (TCN)
Inhibition buffer: 25 mM Tris, 150 mM NaCl, pH 7.5
Assay buffer: 50 mM CHES, 250 mM NaCl, pH 10.0
Kallikrein-1 Protein, Human (240a.a, HEK293, His) (HY-P73262)
Bacterial Thermolysin
1,10-Phenanthroline
Serpin A4 Protein, Human (HEK293, His) (HY-P71143)
Substrate: Pro-Phe-Arg-7-amido-4-methylcoumarin (PFR-AMC, HY-137784)

Procedure
1. Dilute rhKLK1 to 100 μg/mL in activation buffer (containing 1 μg/mL Bacterial Thermolysin) .
2. Incubate at 37°C for 1 hour.
3. Add 1,10-Phenanthroline to a final concentration of 10 mM to stop the activation reaction.
4. Prepare different concentrations of Human Serpin A4 in assay buffer with the following serial dilutions: 8000, 4000, 2000, 1000, 625, 415, 200, 75 nM.
5. Dilute active Human KLK1 to 5 μg/mL in inhibition buffer.
6. Mix 20 μL of 5 μg/mL Human KLK1 with 20 μL of the Human Serpin A4 dilution series. Include two controls: 20 μL of inhibitor buffer and 20 μL of 5 μg/mL Human KLK1.
7. Incubate at 37°C for 1 hour.
8. Dilute the curve 62.5 times by adding assay buffer to each dilution.
9. Dilute the substrate to 200 μM in assay buffer.
10. Add 50 μL of the diluted mixture to the plate and initiate the reaction by adding 50 μL of 200 μM substrate to the wells. For the blank control group, add 50 μL of assay buffer and 50 μL of substrate.
11. Read in kinetic mode for 5 minutes at excitation wavelength 380 nm and emission wavelength 460 nm.
12. Determine the 50% inhibitory concentration (IC₅₀) of Human Serpin A4 by plotting RFU/min versus concentration and fitting the data with a 4-parameter logistic (4-PL) curve.

Human

HEK293

C-6*His

P29622 (Q21-P427)

5267  [NCBI]

Full Length of Mature Protein

QLHVEHDGESCSNSSHQQILETGEGSPSLKIAPANADFAFRFYYLIASETPGKNIFFSPLSISAAYAMLSLGACSHSRSQILEGLGFNLTELSESDVHRGFQHLLHTLNLPGHGLETRVGSALFLSHNLKFLAKFLNDTMAVYEAKLFHTNFYDTVGTIQLINDHVKKETRGKIVDLVSELKKDVLMVLVNYIYFKALWEKPFISSRTTPKDFYVDENTTVRVPMMLQDQEHHWYLHDRYLPCSVLRMDYKGDATVFFILPNQGKMREIEEVLTPEMLMRWNNLLRKRNFYKKLELHLPKFSISGSYVLDQILPRLGFTDLFSKWADLSGITKQQKLEASKSFHKATLDVDEAGTEAAAATSFAIKFFSAQTNRHILRFNRPFLVVIFSTSTQSVLFLGKVVDPTKP

Approximately 50-80&120-130 kDa, based on SDS-PAGE under reducing conditions, due to the glycosylation.

Greater than 95% as determined by reducing SDS-PAGE.

无菌粉末

1.Lyophilized from a 0.22 μm filtered solution of 50 mM Tris-HCl, 150 mM NaCl, 10 mM NaCl, pH 8.0.
2.Lyophilized from a 0.22 μm filtered solution of 20 mM PB, 6% Trehalose, 4% Mannitol, 50 mM NaCl, 0.05% Tween 80, pH 6.0.
3.Lyophilized from a 0.22 μm filtered solution of 20 mM PB, 50 mM NaCl, pH 6.0, 8% trehalose.
Note: For SPR assay, please replace the buffer. Primary amine components (e.g., Tris, imidazole) can affect protein-coupled chips.

<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.