Recombinant Human Complement Factor D Protein, CF Summary
Ile26-Ala253, 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, NaCl, CaCl2 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.
- 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
- Dilute rhFactor D to 5 ng/µL in Assay Buffer.
- Dilute Substrate to 200 µM in Assay Buffer with 200 µM DTNB.
- 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.
- Read in kinetic mode for 20 minutes at an absorbance of 405 nm.
- Calculate specific activity:
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 0.320 cm
Note: the output of many spectrophotometers is in mOD Per Well:
- rhFactor D: 0.25 µg
- DTNB: 100 µM
- Substrate: 100 µM
Background: Complement Factor D/Adipsin
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).
- White, R.T. et al. (1992) J. Biol. Chem. 267:9210.
- Taylor, F.R. et al. (1999) Biochemistry 38:2849.
- Kim, S. et al. (1995) J. Biol. Chem. 270:24399.
Citations for Recombinant Human Complement Factor D Protein, CF
R&D Systems personnel manually curate a database that contains references using R&D Systems products. The data collected includes not only links to publications in PubMed, but also provides information about sample types, species, and experimental conditions.
Citations: Showing 1 - 2
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Adiponectin inhibits insulin function in primary trophoblasts by PPARalpha-mediated ceramide synthesis.
Authors: Aye I, Gao X, Weintraub S, Jansson T, Powell T
Mol Endocrinol, 2014;28(4):512-24.
Sample Types: Whole Cells
A multiplex immunoassay for human adipokine profiling.
Authors: Schipper HS, De Jager W, van Dijk ME, Meerding J, Zelissen PM, Adan RA, Prakken BJ, Kalkhoven E
Clin. Chem., 2010;56(0):1320.
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