Recombinant Mouse Complement Component C1ra Protein, CF

• Reactivity: Mu
• Applications: Enzyme Activity
• Format: Carrier-Free

Recombinant Mouse Complement Component C1ra Protein, CF Summary

• Details of Functionality: 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: Ser17 and Ile463
• Protein/Peptide Type: Recombinant Enzymes
• Gene: C1ra
• Purity: >80%, by SDS-PAGE under reducing conditions and visualized by Colloidal Coomassie® Blue stain at 5 μg per lane
• Endotoxin Note: <1.0 EU per 1 μg of the protein by the LAL method.

Applications/Dilutions

• Dilutions:
  • Enzyme Activity
• Theoretical MW: 51 kDa (heavy chain) & 28 kDa (light chain).
  Disclaimer note: The observed molecular weight of the protein may vary from the listed predicted molecular weight due to post translational modifications, post translation cleavages, relative charges, and other experimental factors.
• SDS-PAGE: 58-59 kDa and 35-36 kDa, reducing conditions

Packaging, Storage & Formulations

• 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.
• Buffer: Supplied as a 0.2 μm filtered solution in Tris, CaCl, NaCl, and Glycerol.
• Purity: >80%, by SDS-PAGE under reducing conditions and visualized by Colloidal Coomassie® Blue stain at 5 μg per lane
• Assay Procedure:
  • 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
  1. Dilute rmC1Ra to 0.5 ng/µL in Assay Buffer.
  2. Dilute Substrate to 200 µM in Assay Buffer with 200 µM of DTNB.
  3. 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.
  4. Read in kinetic mode for 5 minutes at an absorbance of 405 nm.
  5. Calculate specific activity:

  Specific Activity (pmol/min/µg) = (Adjusted Vmax* (OD/min) x well volume (L) x 10^12 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

Notes

Coomassie is a registered trademark of Imperial Chemical Industries Ltd.


This product is produced by and ships from R&D Systems, Inc., a Bio-Techne brand.

Alternate Names for Recombinant Mouse Complement Component C1ra Protein, CF

• C1r;Complement C1r-A subcomponent;mC1;mC1rA;Peptidase S1 domain-containing protein
• C1ra
• Complement Component C1ra

Background

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.

1. Arlaud, G. J. et al. (2002) Biochem. Soc. Trans. 30:1001.
2. Lacroix, M. et al. (2001) J. Biol. Chem. 276:36233.
3. Garnier, G. et al. (2003) J. Biochem. 371:630.

Bioinformatics

• Gene Symbol: C1ra
• Uniprot:
  • AAH04637()