Recombinant Human Complement Factor D Protein, CF

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

Recombinant Human Complement Factor D Protein, CF Summary

• Details of Functionality: Measured by its ability to cleave a colorimetric peptide substrate, N-carbobenzyloxy-Lys-ThioBenzyl ester (Z-Lys-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 >70 pmol/min/µg, as measured under the described conditions.
• Source: Mouse myeloma cell line, NS0-derived human Complement Factor D/Adipsin protein
  Ile26-Ala253, with a C-terminal 10-His tag
• Accession #: P00746
• N-terminal Sequence: Ile26 & His35
• Protein/Peptide Type: Recombinant Enzymes
• Gene: CFD
• Purity: >95%, by SDS-PAGE visualized with Silver Staining and quantitative densitometry by Coomassie® Blue Staining.
• Endotoxin Note: <1.0 EU per 1 μg of the protein by the LAL method.

Applications/Dilutions

• Dilutions:
  • Enzyme Activity
• Theoretical MW: 26 kDa & 25 kDa.
  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: 27 kDa & 26 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, NaCl, CaCl₂ and Glycerol.
• Purity: >95%, by SDS-PAGE visualized with Silver Staining and quantitative densitometry by Coomassie® Blue Staining.
• Assay Procedure:
  • Assay Buffer: 50 mM Tris, 1 M NaCl, pH 7.5
  • Recombinant Human Complement Factor D/Adipsin (rhFactor D) (Catalog # 1824-SE)
  • Substrate: Z-Lys-SBzl (Bachem, Catalog # M-1300), 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 rhFactor D to 5 ng/µL in Assay Buffer.
  2. Dilute Substrate to 200 µM in Assay Buffer with 200 µM DTNB.
  3. Load 50 µL of the diluted rhFactor D into a clear plate, and start the reaction by adding 50 µL of Substrate/DTNB mixture to wells. Include a Substrate Blank containing 50 µL Assay Buffer and 50 µL Substrate mixture without any rhFactor D.
  4. Read in kinetic mode for 20 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 0.320 cm
  Note: the output of many spectrophotometers is in mOD Per Well:
  • rhFactor D: 0.25 µg
  • DTNB: 100 µM
  • Substrate: 100 µM

Notes

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

Alternate Names for Recombinant Human Complement Factor D Protein, CF

• Adipsin
• ADN
• ADNcomplement factor D
• AMBP-1
• C3 convertase activator
• CFD
• complement factor D (adipsin)
• complement factor D preproprotein
• Complement Factor D
• D component of complement (adipsin)
• DF
• EC 3.4.21
• EC 3.4.21.46
• PFD
• Properdin factor DADIPSIN

Background

Complement Factor D is a serine protease that catalyzes the initial proteolytic step in the alternative pathway of complement. Expressed in adipose tissue at high levels, factor D is also known as adipsin (1). It is an exceptionally specific protease and the only known protein substrate is factor B in complex with C3 (2). Factor D protease activity is regulated by reversible conformational changes, which differs from the majority of serine proteases whose regulation involves either activation by processing of the zymogens or inactivation by binding of the inhibitors. Compared to its physiologically important proteolytic activity, factor D has much lower activity toward synthetic peptide substrates. However, thioester substrates have been routinely used for assessing factor D activity (3).

1. White, R.T. et al. (1992) J. Biol. Chem. 267:9210.
2. Taylor, F.R. et al. (1999) Biochemistry 38:2849.
3. Kim, S. et al. (1995) J. Biol. Chem. 270:24399.

Publications for Complement Factor D/Adipsin (1824-SE)(3)

Publications using 1824-SE

• Byeon, HJ;Chae, MK;Ko, J;Lee, EJ;Kikkawa, DO;Jang, SY;Yoon, JS; The Role of Adipsin, Complement Factor D, in the Pathogenesis of Graves' Orbitopathy Investigative ophthalmology & visual science 2023-08-01 [PMID: 37555734] (Bioassay, Human)
• Aye I, Gao X, Weintraub S, Jansson T, Powell T Adiponectin inhibits insulin function in primary trophoblasts by PPARalpha-mediated ceramide synthesis. Mol Endocrinol, 2014-02-25;28(4):512-24. 2014-02-25 [PMID: 24606127] (Bioassay, Human)
• Schipper HS, De Jager W, van Dijk ME, Meerding J, Zelissen PM, Adan RA, Prakken BJ, Kalkhoven E A multiplex immunoassay for human adipokine profiling. Clin. Chem., 2010-06-08;56(0):1320. 2010-06-08 [PMID: 20530731] (ELISA (Standard))

Bioinformatics

• Gene Symbol: CFD
• Uniprot:
  • P00746()