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- Regulatory Status
- RUO
- Other Names
- DF, ADN, PFD, Adipsin, Properdin Factor D, C3 Convertase Activator, Complement Factor D (Adipsin)
- Ave. Rating
- Submit a Review
- Product Citations
- publications
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Recombinant human complement factor D activity is 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). The specific activity is > 70 pmol/min/µg. -
Stability Testing for Recombinant Human Complement Factor D. Recombinant human complement factor D was aliquoted in PBS pH 7.2. One aliquot was frozen and thawed four times (4x Freeze/Thaw) and compared to the control that was kept at 4°C (Control). The samples were tested for their 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). The specific activity is > 70 pmol/min/µg.
Complement Factor D (Adipsin) is a serine protease with restricted substrate specificity. It cleaves a single lysine-arginine bond in complement factor B, only when B is complexed with complement component C3 (C3b-bound factor B). Factor D limits the rate of the alternative complement pathway. Adipose tissue is the main source of factor D. Also, monocytes and macrophages express this protein. Studies in Masp1/3−/− mice (Mannose-binding lectin-associated serine protease) identified that factor D is produced as a pro-factor D with the activation peptide QPRGR at its N terminus. Masp1/3−/− mice do not show alternative complement pathway activation in serum; suggesting that Masp1/3−/− mice do not change pro-factor D to its active form. Adipocytes produce complement component 3, and complement factors B and D. Factor D induces adipocyte differentiation through the induction of C3a and C3aR, thus the interaction between C3a and C3aR induces triglyceride synthesis. Studies with Adipsin -/- mice suggest that factor D improves β cell function in diabetes. Factor D is associated to the onset of pulmonary arterial hypertension in systemic sclerosis. In addition, factor D seems to be a prognostic biomarker in patients with coronary artery diseases.
Product DetailsProduct Details
- Source
- Human Complement Factor D, amino acid Ile26-Ala253 (Accession #: P00746) was expressed in CHO cells. The C-terminus contains 8His- GGQ tag.
- Molecular Mass
- The 241 amino acid recombinant protein has a predicted molecular mass of approximately 25.9 kD. The DTT-reduced and non reduced proteins migrate at approximately 28 kD by SDS-PAGE. The predicted N-terminal amino acid is Ile.
- Purity
- > 95%, as determined by Coomassie stained SDS-PAGE
- Formulation
- 0.22 µm filtered protein solution is in PBS, pH 7.2
- Endotoxin Level
- Less than 0.1 EU per µg cytokine as determined by the LAL method
- Concentration
- 10 and 25 µg sizes are bottled at 200 µg/mL. 100 µg size and larger sizes are lot-specific and bottled at the concentration indicated on the vial. To obtain lot-specific concentration and expiration, please enter the lot number in our Certificate of Analysis online tool.
- Storage & Handling
- Unopened vial can be stored at -20°C or -70°C for six months. For maximum results, quick spin vial prior to opening. Avoid repeated freeze/thaw cycles.
- Activity
- Complement factor D cleaves a colorimetric peptide substrate, N-carbobenzyloxy-Lys-Thiobenzyl ester (Z-Lys-SBzl), in the presence of 5,5’Dithio-bis (2-nitrobenzoic acid) (DTNB). The specific activity is > 70 pmol/min/µg, as measured under the described conditions.
- Application
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Bioassay
- Application Notes
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Assay Procedure
Recombinant Human complement Factor D/Adipsin activity is 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). The increase of the product is monitored by increase in intensity of absorbance at 405 nm.
Materials- Assay buffer: 50 mM Tris, 1 M NaCl, pH 7.5
- Recombinant human Complement Factor D/Adipsin
- Substrate: Z-Lys-SBzl (Bachem, Cat. No. M-1300), 10 mM stock in DMSO
- 5,5’Dithio-bis-(2-nitrobenzoic acid) (DTNB) (Sigma, Cat. No. D-8130), 10 mM stock in DMSO
- 96-well clear plate (Costar, Cat. No. 92592)
- Plate reader (Model: SpectraMax Plus by Molecular Devices) or equivalent
Procedure- Dilute the recombinant factor D to 5 ng/µL in assay buffer.
- Dilute the substrate to 200 µM in assay buffer with 200 µM DTNB.
- Load 50 µL of the diluted recombinant factor D into a clear plate, and start the reaction by adding 50 µL of substrate/DTNB mixture to the wells. Include a substrate blank containing 50 µL assay buffer and 50 µL substrate mixture without any recombinant Factor D.
- Read in kinetic mode for 5 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- Recombinant factor D: 0.25 µg
- DTNB: 100 µM
- Substrate: 100 µM
BioLegend carrier-free recombinant proteins provided in liquid format are shipped on blue-ice. Our comparison testing data indicates that when handled and stored as recommended, the liquid format has equal or better stability and shelf-life compared to commercially available lyophilized proteins after reconstitution. Our liquid proteins are verified in-house to maintain activity after shipping on blue ice and are backed by our 100% satisfaction guarantee. If you have any concerns, contact us at tech@biolegend.com.
Antigen Details
- Structure
- Monomer
- Distribution
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Adipose tissue, monocytes, macrophages, microvascular endothelial cells
- Function
- Alternative pathway of the complement system, cleaves complement factor B, promotes adipocyte differentiation
- Ligand/Receptor
- Complement factor B
- Bioactivity
- Complement factor D cleaves a colorimetric peptide substrate, N-carbobenzyloxy-Lys-Thiobenzyl ester (Z-Lys-SBzl), in the presence of 5,5’Dithio-bis (2-nitrobenzoic acid) (DTNB).
- Cell Type
- Endothelial cells, Macrophages, Monocytes
- Biology Area
- Cardiovascular Biology, Cell Proliferation and Viability, Complement, Immunology, Innate Immunity
- Molecular Family
- Enzymes and Regulators, Innate Immune Signaling, Proteases
- Antigen References
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- White RT, et al. 1992. J Biol Chem. 267:9210.
- Pattrick M, et al. 2009. Mol Immunol. 46:755.
- Mamane Y, et al. 2009. Diabetes. 58:2006.
- Lo JC, et al. 2014. Cell. 158:41.
- Song NJ, et al. 2016. PLoS One. 11:e0162228.
- Korman BD, et al. 2017. Arthritis Rheumatol. 69:2062.
- Ohtsuki T, et al. 2019. J Am Heart Assoc. 8:e013716.
- Gene ID
- 1675 View all products for this Gene ID
- UniProt
- View information about Complement Factor D on UniProt.org
Related FAQs
- Why choose BioLegend recombinant proteins?
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• Each lot of product is quality-tested for bioactivity as indicated on the data sheet.
• Greater than 95% Purity or higher, tested on every lot of product.
• 100% Satisfaction Guarantee for quality performance, stability, and consistency.
• Ready-to-use liquid format saves time and reduces challenges associated with reconstitution.
• Bulk and customization available. Contact us.
• Learn more about our Recombinant Proteins. - How does the activity of your recombinant proteins compare to competitors?
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We quality control each and every lot of recombinant protein. Not only do we check its bioactivity, but we also compare it against other commercially available recombinant proteins. We make sure each recombinant protein’s activity is at least as good as or better than the competition’s. In order to provide you with the best possible product, we ensure that our testing process is rigorous and thorough. If you’re curious and eager to make the switch to BioLegend recombinants, contact your sales representative today!
- What is the specific activity or ED50 of my recombinant protein?
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The specific activity range of the protein is indicated on the product datasheets. Because the exact activity values on a per unit basis can largely fluctuate depending on a number of factors, including the nature of the assay, cell density, age of cells/passage number, culture media used, and end user technique, the specific activity is best defined as a range and we guarantee the specific activity of all our lots will be within the range indicated on the datasheet. Please note this only applies to recombinants labeled for use in bioassays. ELISA standard recombinant proteins are not recommended for bioassay usage as they are not tested for these applications.
- Have your recombinants been tested for stability?
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Our testing shows that the recombinant proteins are able to withstand room temperature for a week without losing activity. In addition the recombinant proteins were also found to withstand four cycles of freeze and thaw without losing activity.
- Does specific activity of a recombinant protein vary between lots?
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Specific activity will vary for each lot and for the type of experiment that is done to validate it, but all passed lots will have activity within the established ED50 range for the product and we guarantee that our products will have lot-to-lot consistency. Please conduct an experiment-specific validation to find the optimal ED50 for your system.
- How do you convert activity as an ED50 in ng/ml to a specific activity in Units/mg?
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Use formula Specific activity (Units/mg) = 10^6/ ED50 (ng/mL)
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