Recombinant Mouse Complement Component C1ra Protein, CF

Catalog #: 7160-SE Datasheet / COA / SDS

Catalog #	Availability	Size / Price	Qty
7160-SE-010

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Recombinant Mouse Complement Component C1ra Protein, CF Summary

Product Specifications

Purity

>80%, 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 cleave a colorimetric peptide substrate, N-carbobenzyloxy-Gly-Arg-ThioBenzyl ester (Z-GR-SBzl), in the presence of 5,5’Dithio-bis (2-nitrobenzoic acid) (DTNB). Edwards, K.M. et al. (1999) J. Biol. Chem. 274:30468. The specific activity is >5,500 pmol/min/μg, as measured under the described conditions.

Source

Mouse myeloma cell line, NS0-derived mouse Complement Component C1ra protein
Met1-Asn707, with a C-terminal 6-His tag

Accession #

AAH04637

N-terminal Sequence
Analysis

Ser17 and Ile463

Predicted Molecular Mass

51 kDa (heavy chain) & 28 kDa (light chain)

SDS-PAGE

58-59 kDa and 35-36 kDa, reducing conditions

Product Datasheets

7160-SE

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.

7160-SE

Formulation	Supplied as a 0.2 μm filtered solution in Tris, CaCl, NaCl, and Glycerol.
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, pH 7.5
Recombinant Mouse Complement Component C1Ra (rmC1Ra) (Catalog # 7160-SE)
Substrate: Z-Gly-Arg-SBzl (MP Biomedicals, Catalog # SB007), 10 mM stock in DMSO
5,5’Dithio-bis (2-nitrobenzoic acid) (DTNB) (Sigma, Catalog # D-8130), 10 mM stock in DMSO
96 well Clear Plate (Costar, Catalog # 92592)
Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent

Dilute rmC1Ra to 0.5 ng/µL in Assay Buffer.
Dilute Substrate to 200 µM in Assay Buffer with 200 µM of DTNB.
Load 50 µL of the diluted rhC1r into a clear plate, and start the reaction by adding 50 µL of the Substrate/DTNB mixture to wells. Include a Substrate Blank containing 50 µL Assay Buffer and 50 µL Substrate Mixture without any rmC1Ra.
Read in kinetic mode for 5 minutes at an absorbance of 405 nm.
Calculate specific activity:

Specific Activity (pmol/min/µg) =	Adjusted V_max* (OD/min) x well volume (L) x 10¹² pmol/mol
	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:

rmC1Ra: 0.025 μg
DTNB: 100 µM
Substrate: 100 µM

Reconstitution Calculator

Background: Complement Component C1ra

The classical complement pathway plays a major role in innate immunity against infection. This pathway is triggered by C1, a multimolecular complex composed of the recognition protein C1q and two serine proteases, C1r and C1s. Following the C1q recognition, C1r is autoactivated, and in turn activates C1s, which cleaves C4 and C2, the C1 substrates (1). Both C1r and C1s activation involve cleavage of a specific Arg-Ile bond, converting the single-chain proenzymes into active proteases composed of disulfide-linked heavy and light chains (2). The heavy chain contains multiple domains in the order of CUB1-EGF-CUB2-CCP1-CCP2-Activation Peptide. The light chain contains the serine protease catalytic domain. The mouse C1r gene is duplicated as C1rA and C1rB. The C1rA gene is primarily expressed in liver and is therefore the homologue of human C1r gene (3). The full-length mouse C1r-A was expressed and purified from conditioned medium. The purified recombinant mC1rA protein corresponds to the processed active form, with the A and B chains beginning at residues Ser 17 and Ile 463, respectively.

References