Creative Biolabs' more than 10 years of CRO experience gives you the confidence to find the right partner in the complement field, offering an extension to your lab with services and products tailored to your needs. We are here to provide you with key analytical insights to move your research forward. Here, we summarize the C1q fragments expression and purification protocol, hoping to provide a reference to our customers' research.
C1q Fragments Expression and Purification Protocol
Fig.1 Flow chart of the expression and purification C1q fragments protocol. (Creative Biolabs)
Published Data
Fig.2 Purification of mouse C1q and characterization of new monoclonal antibodies.1
Mouse serum C1q was purified through IgG affinity chromatography and subsequent cation exchange, yielding a double peak. The second peak, devoid of aggregates, was verified as pure by SDS-PAGE analysis. Two fusions yielded six hybridoma clones producing C1q-specific antibodies, five being IgM monoclonal antibodies. Clones 9H10 and 2F6, showing strong ELISA signals, were further characterized. Using sandwich ELISA, 9H10 (IgG2b) demonstrated cross-reactivity with C1q from mouse, rat, and human sources, while 2F6 (IgM) was specific to mouse and rat C1q, showing no reactivity with human C1q. In hemolysis assays, 9H10 showed no inhibitory activity, while 2F6 selectively inhibited hemolysis in rat serum. Western blot analysis revealed that 9H10 mainly targeted the C1q A chain, while 2F6 exhibited weak binding, indicating a conformation-dependent epitope. Immunoaffinity chromatography with 9H10 efficiently isolated functional C1q from mouse serum, restoring activity to C1q KO mouse serum.
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Reference
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Byrne, Robert AJ, et al. "Novel monoclonal antibodies against mouse C1q: characterization and development of a quantitative ELISA for mouse C1q." Molecular Neurobiology 58.9 (2021): 4323-4336. Distributed under Open Access license CC BY 4.0, without modification.
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