Recombinant Mouse Serpin A1a Protein, CF

Catalog #: 7690-PI Datasheet / COA / SDS

Catalog #	Availability	Size / Price	Qty
7690-PI-010

R&D Systems Recombinant Proteins and Enzymes

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Recombinant Mouse Serpin A1a Protein, CF Summary

Product Specifications

Purity

>95%, by SDS-PAGE under reducing conditions and visualized by Colloidal Coomassie® Blue stain at 5 μg per lane

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 is <5 nM, as measured under the described conditions.

Source

Mouse myeloma cell line, NS0-derived mouse Serpin A1a protein
Glu25-Lys413, with a C-terminal 6-His tag

Accession #

AAH11040

N-terminal Sequence
Analysis

Glu25

Predicted Molecular Mass

44 kDa

SDS-PAGE

50-60 kDa, reducing conditions

Product Datasheets

7690-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.

7690-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. 6 months from date of receipt, -20 to -70 °C as supplied. 3 months, -20 to -70 °C under sterile conditions after opening.

Assay Procedure

Materials

Assay Buffer: 50 mM Tris, 10 mM CaCl₂, 150 mM NaCl, 0.05% Brij-35 (w/v), pH 7.5 (TCNB)
Trypsin Buffer: 50 mM Tris, 10 mM CaCl₂, 150 mM NaCl, pH 7.5 (TCN)
Recombinant Mouse Serpin A1a (rmSerpin A1a) (Catalog # 7690-PI)
Trypsin, from bovine pancreas (Sigma, Catalog # T1426)
Substrate: MCA-RPKPVE-NVal-WRK(DNP)-NH₂ (Catalog # ES002), 2 mM stock in DMSO
F16 Black Maxisorp Plate (Nunc, Catalog # 475515)
Fluorescent Plate Reader (Model: SpectraMax Gemini EM by Molecular Devices) or equivalent

Prepare a curve of rmSerpin A1a (MW 44,000 Da). Make the following serial dilutions in Assay Buffer: 3600, 1800, 900, 450, 225, 112.5, 56.25, 28.13, and 14.1 nM.
Dilute Trypsin to 5.0 µg/mL in Trypsin Buffer.
Combine equal volumes of dilute rmSerpin A1a and Trypsin. Include a control containing equal volumes of Assay Buffer and Trypsin.
Incubate reactions at room temperature for 30 minutes.
Dilute incubated reaction mixtures 25-fold in Assay Buffer.
Dilute Substrate to 20 µM in Assay Buffer.
Load 50 µL of the incubated reactions into the wells of a black well plate, and start the reaction by adding 50 µL of 20 µM 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% inhibiting concentration of rmSerpin A1a (IC₅₀) by plotting RFU/min (or specific activity) vs. concentration with 4-PL fitting.
The specific activity for Trypsin at each point may be determined using the following formula (if needed):

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

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

Per Well:

Trypsin: 0.005 µg (2 nM)
rmSerpin A1a: 36, 18, 9, 4.5, 2.25, 1.125, 0.563, 0.281, and 0.141 nM
ES002 : 10 µM

Reconstitution Calculator

Background: Serpin A1a

Serpin A1a, also known as alpha-1-antitrypsin, belongs to the serpin superfamily of proteinase inhibitors. It is one of five alpha 1-proteinase inhibitors produced in mice. Serpin A1a is produced in the liver, then secreted into the bloodstream. It inhibits a number of serine proteases, including elastase, trypsin, plasmin, and thrombin (1, 2). Serpin A1a’s major function is to protect the lungs from neutrophil elastase. Serpin A1a deficiency can cause complications such as emphysema, and chronic obstructive pulmonary disease (COPD).

References