Recombinant Mouse Complement Factor D Protein, CF Summary
Ile26-Ser259, with a C-terminal 10-His tag
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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.
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.
|Formulation||Supplied as a 0.2 μm filtered solution in Tris and NaCl.|
|Shipping||The product is shipped with dry ice or equivalent. Upon receipt, store it immediately at the temperature recommended below.|
|Stability & Storage:||Use a manual defrost freezer and avoid repeated freeze-thaw cycles.
- Assay Buffer: 50 mM Tris, 1 M NaCl, pH 7.5
- Recombinant Mouse Complement Factor D/Adipsin (rmFactor D) (Catalog # 9207-SE)
- Substrate: Z-Lys-SBzl (Bachem, Catalog # M-1300), 10 mM stock in DMSO
- 5',5'-Dithiobis(2-nitrobenzoic acid) (DTNB) (Sigma, Catalog # D8130), 10 mM stock in DMSO
- 96-well Clear Plate (Catalog # DY990)
- Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent
- Dilute rmFactor D to 20 µg/mL in Assay Buffer.
- Dilute substrate to 400 µM in Assay Buffer containing 400 µM DTNB.
- Load 50 µL of 20 µg/mL rmFactor D to plate, and start the reaction by adding 50 µL of substrate/DTNB mixture. Include a Substrate Blank containing 50 µL of Assay Buffer and 50 µL of substrate/DTNB mixture.
- Read in kinetic mode for 20 minutes at an absorbance of 405 nm.
Specific Activity (pmol/min/µg) =
|Adjusted Vmax* (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.
- rmFactor D: 1.0 µg
- Substrate: 200 µM
- DTNB: 200 µM
Background: Complement Factor D/Adipsin
Complement Factor D, also known as Adipsin, is a serine protease that catalyzes the initial proteolytic step in the alternative pathway of complement. It cleaves the C3b-bound Factor B, resulting in the formation of a C3bBcomplex, which functions as the alternative pathway C3 convertase (1-3). Factor D is primarily expressed in adipose tissue and is released into the circulation as a zymogen with a 5 amino acid (aa) N-terminal activation peptide (4-6). It is activated by MASP-1 mediated proteolytic removal of the activation peptide (6). Factor D triggered complement activation is important for the clearance of cell debris and control of inflammation following tissue damage (7). The C3 convertase generates C3a which binds to C3a R on pancreatic beta cells to induce insulin production and regulate glucose homeostasis (5). Mature mouse Complement Factor D shares 65% and 83% amino acid sequence identity with human and rat Complement Factor D, respectively. Its protease activity is regulated by reversible conformational changes, which is distinct from most serine proteases whose regulation involves either activation by zymogen processing or inactivation by binding to inhibitors. Compared to its cleavage of C3b-bound Factor B, Factor D has much lower activity toward synthetic peptide substrates. However, thioester substrates have been routinely used for assessing Factor D activity (8).
- Kijlstra, A. and T.T. Berendschot (2015) Ophthalmic Res. 54:64.
- Taylor, F.R. et al. (1999) Biochemistry 38:2849.
- Ricklin, D. et al. (2010) Nat. Immunol. 11:785.
- White, R.T. et al. (1992) J. Biol. Chem. 267:9210.
- Lo, J.C. et al. (2014) Cell 158:41.
- Takahashi, M. et al. (2010) J. Exp. Med. 207:29.
- Cresci, G.A. et al. (2015) Mol. Immunol. 64:9.
- Kim, S. et al. (1995) J. Biol. Chem. 270:24399.
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