Recombinant Mouse Complement Factor D Protein, CF

Catalog #: 9207-SE Datasheet / COA / SDS

Catalog #	Availability	Size / Price	Qty
9207-SE-050

R&D Systems Recombinant Proteins and Enzymes

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All Complement Factor D/Adipsin Proteins and Enzymes

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Summary Product Datasheets Carrier Free Data Images Reconstitution Calculator Background Related Research Areas

Recombinant Mouse Complement Factor D Protein, CF Summary

Product Specifications

Purity

>95%, by SDS-PAGE visualized with Silver Staining and quantitative densitometry by Coomassie® Blue Staining.

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-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 >18 pmol/min/μg, as measured under the described conditions.

Source

Mouse myeloma cell line, NS0-derived mouse Complement Factor D/Adipsin protein
Ile26-Ser259, with a C-terminal 10-His tag

Accession #

P03953

N-terminal Sequence
Analysis

Ile26 and His35

Predicted Molecular Mass

27 kDa

SDS-PAGE

40-49 kDa, reducing conditions

Product Datasheets

9207-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.

9207-SE

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. 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, 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 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:

rmFactor D: 1.0 µg
Substrate: 200 µM
DTNB: 200 µM

Reconstitution Calculator

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).

References

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.

Entrez Gene IDs

1675 (Human); 11537 (Mouse); 54249 (Rat)

Alternate Names

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