Recombinant Mouse Serpin A3N Protein, CF Summary
Product Specifications
Phe21-Lys418, with a C-terminal 6-His tag
Analysis
Product Datasheets
Carrier Free
CF stands for Carrier Free (CF). We typically add Bovine Serum Albumin (BSA) as a carrier protein to our recombinant proteins. Adding a carrier protein enhances protein stability, increases shelf-life, and allows the recombinant protein to be stored at a more dilute concentration. The carrier free version does not contain BSA.
In general, we advise purchasing the recombinant protein with BSA for use in cell or tissue culture, or as an ELISA standard. In contrast, the carrier free protein is recommended for applications, in which the presence of BSA could interfere.
4709-PI
| Formulation | Supplied as a 0.2 μm filtered solution in Tris and NaCl. |
| Shipping | The product is shipped with polar packs. Upon receipt, store it immediately at the temperature recommended below. |
| Stability & Storage: | Use a manual defrost freezer and avoid repeated freeze-thaw cycles.
|
Assay Procedure
- Assay Buffer: 50 mM Tris, pH 7.5
- Activation Buffer: 50 mM MES, 50 mM NaCl, pH 5.5
- Recombinant Mouse Serpin A3N (rmSerpin A3N) (Catalog # 4709-PI)
- Recombinant Human Granzyme B (rhGranzyme B) (Catalog # 2906-SE)
- Recombinant Mouse Active Cathepsin C/DPPI (rmCathepsin C) (Catalog # 2336-CY)
- E-64 (Sigma, Catalog # E-3132), 1 mM stock in DMSO
- DTNB (5,5’-dithio-bis (2-nitrobenzoic acid) (Sigma, Catalog # D-8130), 10 mM stock in DMSO
- Substrate: tert-Butoxycarbonyl-Ala-Ala-Asp-thiobenzyl ester (SM Biochemicals LLC, Catalog # SMSB05), 10 mM stock in DMSO
- 96-well Clear Plate (Costar, Catalog # 92592)
- Plate reader (Model: SpectraMax Plus by Molecular Devices) or equivalent
- Activate rhGranzyme B by diluting to 100 µg/mL with 10 µg/mL of rmCathepsin C in Activation Buffer.
- Incubate at 37 °C for 4 hours.
- Stop reaction with E-64 at final a concentration of 10 µM in Activation Buffer.
- Prepare a curve of rmSerpin A3N (MW: 45551 Da) in Assay Buffer. Make the following serial dilutions: 5000, 2500, 1000, 800, 650, 500, 250, 125, 50, and 10 nM.
- Dilute activated rhGranzyme B to 12.5 µg/mL in Assay Buffer.
- Combine 20 µL of 12.5 µg/mL rhGranzyme B with 20 µL of the rmSerpin A3N serial curve dilutions. Include two enzyme controls of 20 µL of 12.5 µg/mL rhGranzyme B with 20 µL Assay Buffer.
- Incubate mixtures at room temperature for 30 minutes.
- Dilute mixtures by adding 460 µL Assay Buffer to each.
- Dilute Substrate to 200 µM containing 200 µM of DTNB in Assay Buffer.
- In a plate load 50 µL of the diluted mixtures into wells.
- Start the reaction by adding 50 µL of 200 µM Substrate mixture.
- Read at a wavelength of 405 nm in kinetic mode for 5 minutes.
- Derive the 50% inhibiting concentration (IC50) for rmSerpin A3N by plotting OD/min (or specific activity) vs. concentration with 4-PL fitting.
- The specific activity for rhGranzyme B at each point may be determined using the following formula (if needed):
|
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.
Per Well:- rhGranzyme B: 0.025 µg
- rmSerpin A3N curve: 100, 50, 20, 16, 13, 10, 5, 2.5, 1, 0.2, and 0 nM
- Substrate: 100 µM
- DTNB: 100 µM
Reconstitution Calculator
Background: Serpin A3N
Serpin A3N is a serine protease inhibitor that is structurally related to alpha 1-antichymotrypsin encoded by the SERPINA3 gene (1). Serpin A3N is highly expressed in brain, testis, lung, thymus, and spleen (2). Serpin A3N secreted by Sertoli cells may regulate the activity of locally produced Granzyme B (3). Granzyme B inhibition by Serpin A3N may therefore regulate Granzyme B-mediated killing by cytotoxic lymphocytes, providing a means to disable cell-mediated immune responses.
- Forsyth, S. et al. (2003) Genomics 81:336.
- Horvath, A. J. et al. (2004) J. Mol. Evol. 59:488.
- Hirst, C. E. et al. (2001) Mol. Hum. Reprod. 7:1133.
Citations for Recombinant Mouse Serpin A3N Protein, CF
R&D Systems personnel manually curate a database that contains references using R&D Systems products. The data collected includes not only links to publications in PubMed, but also provides information about sample types, species, and experimental conditions.
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Citations: Showing 1 - 4
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Reactive astrocytes transduce inflammation in a blood-brain barrier model through a TNF-STAT3 signaling axis and secretion of alpha 1-antichymotrypsin
Authors: H Kim, K Leng, J Park, AG Sorets, S Kim, A Shostak, RJ Embalabala, K Mlouk, KA Katdare, IVL Rose, SM Sturgeon, EH Neal, Y Ao, S Wang, MV Sofroniew, JM Brunger, DG McMahon, MS Schrag, M Kampmann, ES Lippmann
Nature Communications, 2022-11-02;13(1):6581.
Species: Mouse
Sample Types: Whole Tissue
Applications: Bioassay -
Longitudinal single cell transcriptomics reveals a role for Serpina3n-mediated resolution of inflammation in a mouse colitis model
Authors: YT Ho, T Shimbo, E Wijaya, T Kitayama, S Takaki, K Ikegami, K Miyashita, Y Ouchi, E Takaki, R Yamamoto, Y Kaneda, K Tamai
Cellular and Molecular Gastroenterology and Hepatology, 2021-04-13;0(0):.
Species: Mouse
Sample Types: In Vivo
Applications: Bioassay -
SerpinA3n affects ovalbumin (OVA)-induced asthma in neonatal mice via the regulation of collagen deposition and inflammatory response
Authors: HT Zhang, P Wang, Y Li, MM Zhang
Respiratory physiology & neurobiology, 2021-02-17;288(0):103642.
Species: Mouse
Sample Types: In Vivo
Applications: Bioassay -
The serine protease inhibitor SerpinA3N attenuates neuropathic pain by inhibiting T cell-derived leukocyte elastase.
Authors: Vicuna L, Strochlic D, Latremoliere A, Bali K, Simonetti M, Husainie D, Prokosch S, Riva P, Griffin R, Njoo C, Gehrig S, Mall M, Arnold B, Devor M, Woolf C, Liberles S, Costigan M, Kuner R
Nat Med, 2015-04-27;21(5):518-23.
Species: Mouse
Sample Types: In Vivo, Protein
Applications: Bioassay, In Vivo
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