Recombinant Mouse HAI-2B Protein, CF

Catalog #: 1107-PI Datasheet / COA / SDS

Catalog #	Availability	Size / Price	Qty
1107-PI-010

R&D Systems Recombinant Proteins and Enzymes

1 Image

Product Details

Supplemental Products

Reviews

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

Recombinant Mouse HAI-2B Protein, CF Summary

Product Specifications

Purity

>95%, 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 <2 nM, as measured under the described conditions.

Source

Mouse myeloma cell line, NS0-derived mouse HAI-2B protein
Arg30-Lys140, with a C-terminal 10-His tag

Accession #

NP_001076017

N-terminal Sequence
Analysis

Arg30

Predicted Molecular Mass

14 kDa

SDS-PAGE

23 kDa, reducing conditions

Product Datasheets

1107-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.

1107-PI

Formulation	Lyophilized from a 0.2 μm filtered solution in PBS.
Reconstitution	Reconstitute at 100 μg/mL in sterile, deionized water.
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 reconstitution.

Assay Procedure

Materials

Assay Buffer: 50 mM Tris, 10 mM CaCl2, 150 mM NaCl, 0.05% (w/v) Brij-35, pH 7.5 (TCNB)
Recombinant Mouse HAI-2B (rmHAI-2B) (Catalog # 1107-PI)
Trypsin (Sigma, Catalog # T-1426)
Substrate: MCA-Arg-Pro-Lys-Pro-Val-Glu-NVAL-Trp-Arg-Lys(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

Dilute Trypsin to 0.25 µg/mL in Assay Buffer.
Prepare a curve of rmHAI-2B (MW: 14,281 Da) in Assay Buffer. Make the following serial dilutions: 1000, 200, 50, 25, 12.5, 6.25, 3.13, 1.56, and 0.391 nM.
Mix equal volumes of the rmHAI-2A curve dilutions and 0.25 µg/mL Typsin. Include a control (in duplicate) containing Assay Buffer and 0.25 µg/mL Typsin.
Incubate reactions for 30 minutes at room temperature.
After incubation, dilute the mixtures 5 fold in Assay Buffer.
Dilute Substrate to 20 µM in Assay Buffer.
Load 50 µL of the diluted incubated mixtures into a 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% inhibition concentration (IC₅₀) value for rmHAI-2B by plotting RFU/min (or specific activity) vs. concentration with 4-PL fitting.
The specific activity of 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: 0.00125 µg
rmHAI-2B curve: 50, 10, 2.5, 1.25, 0.625, 0.313, 0.156, 0.078 and 0.0195 nM
Substrate: 10 µM

Reconstitution Calculator

Background: HAI-2B

Three alternatively spliced forms of HAI-2 have been found in mouse tissues (1). HAI-2A, the full-length molecule, consists of two Kunitz domains and a C-terminal transmembrane domain. HAI-2A is a major form expressed in human tissues, but a minor form in mouse tissues. HAI-2B, with residues 37‑93 spliced out and thus lack of the first Kunitz domain, is a major form in mouse tissues. HAI-2B is an effective inhibitor of both HGF activator and trypsin (2).

References