C1R Antibody

What is C1R and why is it important in complement research?

Complement component 1, r subcomponent (C1R) is a critical serine protease within the classical complement pathway. It functions as part of the C1 complex, a calcium-dependent assembly composed of recognition subcomponent C1q (460 kDa) and serine protease subcomponents including two C1r polypeptides (90 kDa) and two C1s polypeptides (80 kDa) . C1r plays a pivotal role in transforming activation signals into enzymatic activity within the complement cascade. When C1q binds to immune complexes or pathogen surfaces, it changes conformation, activating C1r's protease activity, which subsequently cleaves and activates C1s . This activation initiates the downstream classical complement pathway, making C1R antibodies essential tools for studying complement activation, immune responses, and various pathological conditions.

What applications are most suitable for C1R antibody research?

C1R antibodies have demonstrated utility across multiple research applications with validated results. Primary applications include Western Blot (WB) with recommended dilutions ranging from 1:5000-1:50000, Immunohistochemistry (IHC) at dilutions of 1:50-1:500, Immunoprecipitation (IP), and ELISA . Published research has confirmed successful application in all these methodologies, with reactivity primarily in human samples and cited reactivity in mouse models . For optimal experimental design, researchers should consider the specific application requirements and conduct preliminary titration studies to determine ideal antibody concentrations for their particular experimental system.

What sample types can be effectively analyzed using C1R antibodies?

C1R antibodies have demonstrated efficacy across diverse biological sample types. For Western blot applications, human plasma and blood samples show positive detection . In immunohistochemical applications, human liver tissue has been successfully used with recommended antigen retrieval using TE buffer (pH 9.0) or alternatively with citrate buffer (pH 6.0) . For ELISA-based detection methods, validated sample types include plasma, serum, saliva, urine, milk, cerebrospinal fluid (CSF), and cell culture samples . This versatility makes C1R antibodies valuable tools for multi-platform research approaches investigating complement system function across different biological contexts.

How should researchers optimize C1R antibody dilutions for different applications?

Optimization of C1R antibody dilutions is application-dependent and requires methodical titration. For Western Blot applications, a broad dilution range of 1:5000-1:50000 has been validated . Immunohistochemistry applications typically require more concentrated antibody solutions, with recommended dilutions between 1:50-1:500 . For novel experimental systems or non-standard applications, researchers should perform systematic dilution series experiments beginning with manufacturer-recommended ranges and adjusting based on signal-to-noise ratios. The technical data from Proteintech emphasizes that "this reagent should be titrated in each testing system to obtain optimal results" . Sample-dependent factors may necessitate further adjustments, particularly when working with tissues or cell types not previously validated. Researchers should maintain detailed records of optimization experiments to ensure reproducibility in subsequent studies.

What are the critical parameters for designing C1R functional studies in cell culture systems?

When designing functional studies to investigate C1R activity in cell culture systems, several critical parameters must be considered. Based on published methodologies, researchers have successfully employed HEK293T cells cultured in DMEM supplemented with 10% fetal bovine serum (FBS), penicillin/streptomycin, and 2 mM L-glutamine . For transfection experiments, a protocol using 12 μl Turbofect with 4 μg of C1R plasmid in 1 ml EMEM medium (serum-free during transfection) has proven effective . Researchers should consider:

• Cell density optimization: Seeding 1 × 10^6 cells into T-25 flasks the day before transfection

• Medium conditions: Exchange to serum-free medium during transfection, followed by complete medium post-transfection

• Collection timing: Harvesting cells and supernatants 48 hours post-transfection

• For C1r-C1s interaction studies: Mix concentrated supernatants containing C1r (wild-type or variant) 1:1 with supernatant from C1s-transfected cells, incubate for 1 hour at 37°C before western blot analysis

These parameters ensure optimal expression and functional activity assessment of C1R in experimental systems.

What controls should be included when using C1R antibodies for complement activation studies?

Rigorous control implementation is essential for reliable interpretation of C1R antibody-based complement activation studies. Based on methodological approaches in the literature, researchers should include:

• Positive controls:

  • Known C1R-expressing tissues/cells (human plasma, human blood for WB; human liver tissue for IHC)

  • Recombinant C1R protein standards for quantitative assays

• Negative controls:

  • Isotype control antibodies (Rabbit IgG for polyclonal antibodies)

  • C1R-deficient samples when available

  • Secondary antibody-only controls to assess non-specific binding

• Experimental controls:

  • When studying C1r-C1s interactions, include wild-type C1r as reference standard

  • For activation studies, include both activated and non-activated complement samples

  • When examining C1r cleavage activity, compare C1s cleavage patterns between wild-type and variant C1r proteins

• Technical controls:

  • Multiple dilutions of antibody to ensure detection is within linear range

  • Loading controls appropriate to sample type (total protein stains for secreted proteins)

How can C1R antibodies be utilized to investigate structural interactions within the C1 complex?

C1R antibodies are valuable tools for elucidating the complex structural interactions within the C1 complex, particularly the C1r-C1s interaction. Recent structural studies have revealed an extensive interface between C1r and C1s involving their N-terminal regions . Researchers can employ C1R antibodies in several sophisticated approaches:

• Co-immunoprecipitation (Co-IP) studies: Using C1R antibodies to pull down the C1 complex and analyze associated proteins can reveal interaction partners and stoichiometry in native conditions.

• Proximity ligation assays: Combining C1R and C1s antibodies in proximity ligation assays can provide spatial information about their interaction in situ.

• Structural analysis validation: C1R antibodies can be used to validate structural models derived from electron microscopy (EM) or X-ray crystallography. Research has shown that "models of C1 are compatible with negative-stain EM images of cross-linked C1 in which a central mass is visible between the C1q stalks (the CUB1-EGF-CUB2 domains of C1r and C1s and the catalytic domains of C1r)" .

• Domain-specific interaction studies: Using antibodies targeting specific domains of C1r can help map interaction interfaces with C1s and C1q within the assembled C1 complex.

These approaches contribute to understanding the "bouquet-like architecture" of the C1 complex and how conformational changes propagate through the assembly during complement activation .

What methodological approaches can be used to study C1R mutations and their functional consequences?

Investigating C1R mutations and their functional impacts requires integrated methodological approaches. Based on published research protocols, a comprehensive strategy includes:

• Overexpression systems:

  • Cloning wild-type and mutant C1R into expression vectors

  • Transfection into HEK293T cells for protein production

  • Collection of cellular and secreted protein fractions

• Functional activity assessment:

  • Evaluating C1r proteolytic activity by its ability to cleave C1s

  • Mixing supernatants containing C1r (wild-type or variant) with C1s and analyzing cleavage patterns

  • Incubating mixed supernatants at 37°C for 1 hour followed by western blot analysis using C1s antibodies

• Structural integrity analysis:

  • Using western blot to compare expression levels, molecular weights, and cleavage patterns

  • Assessing protein secretion efficiency between wild-type and mutant C1R

• Complement activation evaluation:

  • Measuring downstream complement component activation (C4, C3)

  • Quantifying MAC formation in functional assays

This multi-faceted approach has been successfully employed to study C1R variants associated with periodontal Ehlers-Danlos syndrome (pEDS), revealing how mutations can trigger constitutive complement activation .

How can researchers quantitatively measure C1R activity in clinical and research samples?

Accurate quantification of C1R activity requires appropriate methodological selection based on research objectives. Multiple validated approaches include:

• Enzyme-Linked Immunosorbent Assay (ELISA):

  • Sandwich ELISA techniques using polyclonal antibodies specific for C1r allow detection of C1r in multiple sample types

  • Colorimetric detection provides quantitative results within a detection range of 0.125-8 ng/mL

  • Minimum detection limit: 0.125 ng/mL

  • Sample types: plasma, serum, saliva, urine, milk, CSF, and cell culture samples

• Functional Assays:

  • Hemolysis assays (CH50) measure total complement classical pathway activity

  • Functional ELISAs using coated IgM and colorimetric substrates assess classical pathway activity in serum

  • C1-specific assays through capture of C1q in serum provide better measurement of C1s activity

• Proteomic Approaches:

  • LC-MS/MS methods allow quantification of total C1s protein

  • Gelatin zymography can assess enzymatic activity

• Genetic Analysis:

  • Next-generation sequencing (NGS) and qPCR for studies of congenital complement deficiency or genetic polymorphisms

Researchers should select the most appropriate method based on their specific experimental questions, sample availability, and required sensitivity.

What explains the discrepancy between calculated and observed molecular weights of C1R?

Researchers frequently observe discrepancies between calculated and observed molecular weights of C1R in experimental systems. According to technical data, C1R has a calculated molecular weight of 80 kDa (705 amino acids), but western blot analysis typically reveals observed molecular weights of 85 kDa and 30 kDa . These discrepancies arise from several biological and experimental factors:

• Post-translational modifications: C1r undergoes various modifications including glycosylation, as it is described as "a single-chain glycoprotein" .

• Proteolytic processing: C1r "can be cleaved into an A chain and a B chain upon activation" , explaining the detection of multiple bands. The 85 kDa band likely represents the full-length protein, while the 30 kDa band corresponds to one of the cleavage products.

• Sample preparation conditions: Reducing versus non-reducing conditions can affect observed migration patterns.

• Buffer composition: Salt concentration and pH can influence protein migration in electrophoretic systems.

When interpreting C1R western blot results, researchers should anticipate these molecular weight variations and include appropriate size markers and controls to correctly identify C1R-specific bands.

What strategies can address non-specific binding issues when working with C1R antibodies?

Non-specific binding can compromise data interpretation in C1R antibody applications. To minimize these issues, researchers should implement the following evidence-based strategies:

• Optimize blocking conditions:

  • Use 3-5% BSA or milk proteins in TBS-T for western blots

  • For IHC applications, employ species-appropriate serum or commercial blocking reagents

  • Consider dual blocking with both protein and detergent-based blockers

• Optimize antibody dilutions:

  • Perform titration experiments to determine optimal concentration ranges (WB: 1:5000-1:50000; IHC: 1:50-1:500)

  • More dilute antibody solutions often reduce non-specific binding while maintaining specific signal

• Implement stringent washing protocols:

  • Increase number and duration of wash steps

  • Use appropriate detergent concentration in wash buffers

• Use validated sample preparation methods:

  • For IHC applications, employ recommended antigen retrieval methods: "suggested antigen retrieval with TE buffer pH 9.0; (*) Alternatively, antigen retrieval may be performed with citrate buffer pH 6.0"

  • For western blot, ensure complete denaturation and appropriate sample loading

• Include appropriate controls:

  • Use isotype controls at equivalent concentrations

  • Include competing peptide controls when available

  • Test antibody on known negative samples/tissues

These optimization strategies should be systematically evaluated and documented to establish reliable protocols for specific experimental systems.

How can researchers distinguish between different activation states of C1R in experimental systems?

Distinguishing between inactive (zymogen) and active forms of C1R is crucial for functional studies. Based on current methodologies, researchers can employ several approaches:

• Western blot analysis of cleavage patterns:

  • The inactive C1r zymogen appears as a single-chain glycoprotein

  • Upon activation, C1r is cleaved into A and B chains, detectable as distinct bands

  • Compare patterns between activated and non-activated samples

• Functional enzymatic assays:

  • C1r activation enables C1s cleavage, which can be monitored by mixing C1r-containing supernatants with C1s and analyzing by western blot

  • Active C1r will generate C1s cleavage products not observed with inactive C1r

• Conformation-specific antibodies:

  • Some antibodies preferentially recognize active or inactive conformations

  • Epitope mapping can identify antibodies that differentiate activation states

• Detection of downstream activation products:

  • Active C1r leads to C1s activation, which cleaves C4 and C2, resulting in C3-convertase formation

  • Measuring C4 cleavage products can indirectly confirm C1r activation

• Structural analysis:

  • Active and inactive C1r adopt different conformations within the C1 complex

  • Structural techniques like EM can visualize these conformational differences

By combining these approaches, researchers can reliably distinguish between C1R activation states and characterize functional impacts of mutations or experimental manipulations.

How are C1R antibodies being utilized in therapeutic development research?

C1R antibodies are increasingly employed in therapeutic development research, particularly targeting complement-mediated pathologies. Recent advances in this field include:

• Monoclonal antibody development:

  • Anti-C1s monoclonal antibodies (TNT003, TNT005, TNT009) have demonstrated inhibitory effects on classical pathway activation without affecting alternative and lectin pathways

  • These antibodies have been shown to block C3d deposition in aortic endothelial cells, critical for microvascular inflammation during antibody-mediated rejection (AMR)

• Transplantation research:

  • C1s-targeted approaches have shown promise in preventing complement activation during antibody-mediated rejection in solid organ transplantation

  • Importantly, C1s-specific antibody TNT005 "did not abolish the therapeutic effects of anti-Neisseria meningitidis and Streptococcus pneumoniae antibodies," suggesting pathway-specific inhibition that preserves important immune functions

• Selective pathway inhibition strategies:

  • Targeting C1r/C1s allows specific inhibition of the classical pathway while maintaining alternative and lectin pathway function

  • This selective approach may "reduce the production of auto-antibodies" while preserving important aspects of innate immunity

• Clinical trial progress:

  • Multiple C1s-targeted monoclonal antibodies have entered clinical trials for various indications

  • These developments demonstrate translational potential of C1R research in therapeutic contexts

These applications highlight the evolving role of C1R antibodies from research tools to potential therapeutic agents.

What advanced imaging techniques can be combined with C1R antibodies for structural research?

Integration of C1R antibodies with advanced imaging methodologies enables sophisticated structural analyses of the complement system. Current evidence supports several powerful approaches:

• Cryo-electron microscopy (cryo-EM):

  • C1R antibodies can be used to label specific components within the C1 complex

  • Research has shown that "cryoEM data for C1 [show] images appear to show between six and nine peripheral globular structures (six C1q heads, two C1s catalytic domains, and the central collagen hub and/or density from C1r polypeptides)"

  • Immunogold labeling with C1R antibodies can provide precise localization within these structures

• Super-resolution microscopy:

  • Techniques such as STORM or PALM combined with fluorescently-labeled C1R antibodies allow visualization of complement components below the diffraction limit

  • These approaches can reveal nanoscale organization of C1r within the C1 complex in cellular contexts

• Single-particle analysis:

  • C1R antibodies can aid in particle alignment and classification during image processing

  • This facilitates reconstruction of the "bouquet-like architecture" of the C1 complex

• Correlative light and electron microscopy (CLEM):

  • Combining fluorescently-labeled C1R antibodies with electron microscopy provides contextual cellular information alongside ultrastructural details

  • This approach bridges scales from molecular to cellular levels

These advanced imaging approaches, when combined with specific C1R antibodies, provide unprecedented insights into complement system architecture and dynamics.

How can researchers integrate C1R antibody studies with systems biology approaches?

The integration of C1R antibody-based research with systems biology frameworks represents an emerging frontier. Based on current methodological trends, researchers can implement several strategies:

• Multi-omics integration:

  • Combine C1R antibody-based proteomics with transcriptomics and genomics

  • Methods such as LC-MS/MS for C1R protein quantification can be integrated with NGS approaches

  • This integration provides a comprehensive view of C1R regulation from gene to protein

• Network analysis of complement interactions:

  • Use C1R antibodies to identify interaction partners through immunoprecipitation followed by mass spectrometry

  • Map these interactions within the broader complement and immune system networks

  • Analyze how C1r functions within the "calcium-dependent complex of the 3 subcomponents C1q, C1r, and C1s"

• Computational modeling validation:

  • Experimental data from C1R antibody studies can validate computational models of complement activation

  • These models can predict how mutations or therapeutic interventions might affect system behavior

• In vivo imaging with C1R antibodies:

  • Use fluorescently-labeled C1R antibodies for in vivo imaging of complement activation

  • Correlate with clinical parameters and disease progression

  • These approaches help translate molecular understanding to physiological contexts

• High-throughput screening platforms:

  • Develop antibody-based assays suitable for high-throughput screening of C1R modulators

  • These platforms facilitate discovery of novel therapeutic agents targeting the complement system

Integration of these approaches enables researchers to move beyond reductionist views of C1R function toward understanding its role within the complex, interconnected systems of immunity and inflammation.