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Complement C9 has been studied for its pivotal role in forming the channel part of the membrane attack complex (MAC). Monomeric C9 (mC9) binds the C5b-8 complex to form C5b-9 which catalyzes the polymerization of C9 forming C5b-p9 and transmembrane channels leading to lysis of the target cell. Patients with C9 deficiency suffer from recurrent bacterial infections. Creative Biolabs is a provider of outsourced professional laboratory testing services for drug discovery, catering to small pharmaceutical companies and biotechnology companies. Here we summarize the C9 functional assay protocol for your reference.
C9 Functional Assay Protocol
C9 can be measured in a functional assay using C9-depleted serum obtained by passage over an anti-C9 immunoaffinity column.
Fig.1 Flow chart of C9 functional assay protocol. (Creative Biolabs)
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Fig.2 Serum complement functionality assessed at the stage of C9 activation.1
The terminal complement pathway component C9 transforms into transmembrane pores that damage target cell membranes through polymerization. C9 functional assays assess lytic efficiency through erythrocyte hemolysis alongside fluorescence membrane permeability tests and soluble C9 depletion determined by ELISA. Recent advancements emphasize standardized protocols for maintaining protein stability, including optimized buffer systems to prevent spontaneous C9 activation and thioester hydrolysis. Glycosylation status significantly impacts C9 oligomerization kinetics, as deglycosylation alters hydrodynamic radius and binding avidity, factors quantifiable through SEC-MALS and SDS-PAGE. These methodologies enable precise correlation between structural integrity and functional output, critical for therapeutic discovery.
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Creative Biolabs’ clients at top pharmaceutical, biotechnology, and academic institutions, as well as cutting-edge start-ups, rely on our know-how to get consistent, reliable, high-quality data even on the most difficult projects. If you are interested in our complement services and products, contact our team of experts for consultation on your research request.
References
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Kotimaa, Juha, et al. "Sex matters: systemic complement activity of female C57BL/6J and BALB/cJ mice is limited by serum terminal pathway components." Molecular Immunology 76 (2016): 13-21. Distributed under an Open Access license CC BY 4.0, without modification.
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