Recombinant Mouse Serpin A9/Centerin Protein, CF

Catalog #: 5679-PI Datasheet / COA / SDS

Catalog #	Availability	Size / Price	Qty
5679-PI-010

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Summary Product Datasheets Carrier Free Data Images Reconstitution Calculator Background Related Research Areas

Recombinant Mouse Serpin A9/Centerin Protein, CF Summary

Product Specifications

Purity

>90%, by SDS-PAGE under reducing conditions and visualized by silver stain

Endotoxin Level

<1.0 EU per 1 μg of the protein by the LAL method.

Activity

Measured by its ability to inhibit trypsin cleavage of a fluorogenic peptide substrate, Mca-RPKPVE-Nval-WRK(Dnp)-NH₂ (Catalog # ES002). The IC₅₀ value is <7 nM, measured under the described conditions.

Source

Mouse myeloma cell line, NS0-derived mouse Serpin A9/Centerin protein
Asn26-Met418, with an N-terminal 6-His tag
Accession # Q9D7D2

Accession #

Q9D7D2

N-terminal Sequence
Analysis

His

Structure / Form

Monomer

Predicted Molecular Mass

45 kDa

SDS-PAGE

40-50 kDa, reducing conditions

Product Datasheets

5679-PI

Product Datasheet

COA

SDS

Carrier Free

What does CF mean?

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.

What formulation is right for me?

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.

5679-PI

Formulation	Supplied as a 0.2 μm filtered solution in Tris and NaCl.
Shipping	The product is shipped with dry ice or equivalent. Upon receipt, store it immediately at the temperature recommended below.
Stability & Storage:	Use a manual defrost freezer and avoid repeated freeze-thaw cycles. 6 months from date of receipt, -70 °C as supplied. 3 months, -70 °C under sterile conditions after opening.

Assay Procedure

Materials

Assay Buffer: 50 mM Tris, 10 mM CaCl₂, 0.15 M NaCl, 0.05% (v/v) Brij-35, pH 7.5 (TCNB)
Recombinant Mouse Serpin A9/Centerin (rmSerpin A9) (Catalog # 5679-PI)
Recombinant Human Active Trypsin 3/PRSS3 (rhTrypsin 3) (Catalog # 3714-SE)
Fluorogenic Peptide Substrate: MCA-Arg-Pro-Lys-Pro-Val-Glu-NVAL-Trp-Arg-Lys(DNP)-NH₂ (Catalog # ES002)
F16 Black Maxisorp Plate (Nunc, Catalog # 475515)
Fluorescent Plate Reader (Model: SpectraMax Gemini EM by Molecular Devices) or equivalent

Dilute rhTrypsin 3 to 1 ng/µL in Assay Buffer.
Prepare a curve of rmSerpin A9 (MW: 44,977 Da) in Assay Buffer. Make the following serial dilutions: 1200 nM, 600 nM, 300 nM, 150 nM, 75 nM, 37.5 nM, 18.8 nM, 9.4 nM, 4.7 nM, and 2.3 nM.
Combine equal volumes of dilute rhTrypsin 3 and rmSerpin A9 at each concentration of the curve. Include two controls containing equal volumes of Assay Buffer and diluted rhTrypsin 3 without any rmSerpin A9.
Incubate mixtures at 37 °C for 30 minutes.
Dilute the reaction mixtures 5-fold with Assay Buffer.
Dilute Substrate to 20 µM with Assay Buffer.
Load in a plate 50 µL of the incubated mixtures, and start the reaction by adding 50 µL of 20 mM Substrate.
Read at excitation and emission wavelengths of 320 nm and 405 nm (top read), respectively, in kinetic mode for 5 minutes.
Derive the 50% inhibition concentration (IC₅₀) for rmSerpin A9 by plotting RFU/min (or specific activity) vs concentration with 4-PL fitting.
Calculate specific activity of Trypsin at each point using the following formula (if needed):

Specific Activity (pmoles/min/µg) =	Adjusted V_max* (RFU/min) x Conversion Factor** (pmole/RFU)
	amount of enzyme (µg)

*Adjusted for Substrate Blank

**Derived using calibration standard MCA-Pro-Leu-OH (Bachem, Catalog # M-1975).

Per Well:

rhTrypsin 3: 0.005 µg (2.1 nM)
rmSerpin A9 curve: 60 nM, 30 nM, 15 nM, 7.5 nM, 3.8 nM, 1.9 nM, 0.94 nM, 0.47 nM, 0.23 nM, and 0.12 nM
Substrate: 10 µM

Reconstitution Calculator

Background: Serpin A9/Centerin

Serpin A9, also known as Centerin, is a member of the Serpin superfamily of serine protease inhibitors (1). Serpins are the most broadly distributed superfamily of protease inhibitors. They are known to inhibit serine proteases and some are known to inhibit caspases and papain-like cysteine proteases (1). Recombinant mouse Serpin A9 shows inhibition towards trypsin, thrombin, and plasmin and binds DNA and heparin (2). Its expression is limited to germinal center B cells and lymphoid malignancies with germinal center B-cell maturation (3). New findings suggest that Serpin A9 could be used as a potential diagnostic marker in the recognition of various germinal center derived lymphomas (4).

References