C1RL Antibody

What is C1RL and what are its key structural characteristics?

C1RL, also known as C1r-like serine protease analog protein (CLSPa), is a complement system protein containing a CUB domain and a serine protease domain with a characteristic catalytic triad. The protein plays a role in complement activation and has been implicated in several disease processes .

What are the validated applications for C1RL antibodies in research?

Based on current validation data, C1RL antibodies are suitable for multiple experimental applications:

The antibodies have demonstrated reactivity with human and mouse samples, making them suitable for comparative studies across these species .

How should I validate C1RL antibody specificity for my research?

Proper validation is critical for ensuring reliable results. A methodological approach includes:

• Positive and negative control samples: Include tissues/cells known to express or lack C1RL expression.

• Blocking peptide experiments: Use the immunizing peptide to demonstrate binding specificity.

• siRNA knockdown: Confirm antibody specificity by reducing target protein expression.

• Multiple antibody comparison: Validate results using antibodies targeting different epitopes of C1RL.

When validating polyclonal antibodies like those against C1RL, researchers should verify batch-to-batch consistency, especially when changing lots in long-term studies .

How is C1RL expression altered in cancer, and what are the implications for using C1RL antibodies in cancer research?

Recent studies indicate that C1RL is upregulated in glioblastoma (GBM) and has prognostic value in hepatocellular carcinoma and renal cell cancer . Analysis of 2,120 glioma patients across five public datasets revealed significant correlations between C1RL expression and clinical outcomes, suggesting its potential utility as a biomarker .

When designing studies to investigate C1RL in cancer:

• Use carefully selected tissue microarrays with adequate controls

• Employ standardized scoring systems for immunohistochemistry

• Consider dual immunostaining to analyze C1RL in relation to other cancer markers

• Correlate protein expression with transcriptomic data when available

The altered expression patterns make C1RL a promising target for prognostic studies, but researchers should establish appropriate thresholds for high versus low expression based on their specific experimental context and sample cohort.

What are the optimal experimental conditions for detecting C1RL in different sample types?

Experimental conditions vary by application and sample type:

For Western Blotting:

• Sample preparation should include protease inhibitors to prevent degradation

• For glycosylated C1RL detection, avoid harsh reducing conditions

• Use 8-10% SDS-PAGE gels for optimal separation

• Expected band at 70 kDa (rather than the calculated 53 kDa) due to glycosylation

For Immunohistochemistry/Immunofluorescence:

• Antigen retrieval methods: Heat-induced epitope retrieval in citrate buffer (pH 6.0)

• Blocking: 5-10% normal serum from the same species as the secondary antibody

• Recommended dilution range: 1:50-1:200 for IHC, 0.25-2 μg/mL for IF

• Counterstain: Hematoxylin for IHC; DAPI for nuclear visualization in IF

How can I integrate C1RL antibody data with modern single-cell analysis platforms?

C1RL antibodies can be incorporated into single-cell analysis workflows, particularly with platforms like 10x Genomics' Antibody Capture technology. This approach enables researchers to correlate protein expression with transcriptomic data at the single-cell level .

Key implementation steps:

• Ensure your C1RL antibody is compatible with oligonucleotide conjugation

• Include C1RL in your Feature Reference CSV file with "Antibody Capture" as the feature_type

• Analyze data through visualization tools like Loupe Browser

• Use log-transformed antibody counts for dimensionality reduction analysis

When interpreting results, be aware of potential aggregate formation, which can be identified through metrics provided in the aggregate_barcodes.csv output file from Cell Ranger analysis .

What is known about C1RL's functional role in the complement system and disease processes?

C1RL has been identified as having proteolytic activity within the complement cascade. Research has shown that prohaptoglobin is proteolytically cleaved in the endoplasmic reticulum by C1RL . This activity suggests C1RL plays an important role in protein processing beyond traditional complement activation.

In disease contexts, particularly cancer, C1RL may contribute to immunomodulation through:

• Alteration of complement activation patterns

• Potential involvement in creating immunosuppressive tumor microenvironments

• Interaction with other proteases in the tumor milieu

For functional studies, researchers should consider both gain-of-function approaches (overexpression of C1RL) and loss-of-function studies (siRNA, CRISPR-Cas9) to elucidate its mechanistic contributions to disease processes.

How can I design experiments to explore the relationship between C1RL expression and clinical outcomes?

To effectively study C1RL's prognostic value:

• Patient cohort selection: Include adequate sample sizes with well-documented clinical data and follow-up

• Tissue microarray design: Ensure representation of tumor heterogeneity with multiple cores per patient

• Standardized IHC protocols: Use validated antibodies with consistent staining and scoring systems

• Statistical analysis: Employ Kaplan-Meier survival analysis with multivariate Cox regression to account for confounding factors

Research shows C1RL may have particular relevance in glioma, where immunological and clinicopathological characteristics have been associated with patient outcomes . When designing these studies, consider:

• Stratification by molecular subtypes

• Integration with other established biomarkers

• Correlation with treatment response data when available

What techniques are recommended for studying C1RL interactions with other proteins?

To investigate protein-protein interactions involving C1RL:

• Co-immunoprecipitation: Use C1RL antibodies to pull down protein complexes

• Proximity ligation assay: Visualize and quantify protein interactions in situ

• FRET/BRET: Examine real-time interactions in living cells

• Yeast two-hybrid screening: Identify novel interaction partners

When designing antibody-based interaction studies, consider the epitope location in relation to potential binding domains. The immunogen sequence "NVLPVCLPDNETLYRSGLLGYVSGFGMEMGWLTTELKYSRLPVAPREACNAWLQKRQRPEVFSDNMFCVGDETQ" used for some commercial antibodies should be evaluated to ensure it doesn't interfere with interaction surfaces.

What are common technical challenges when working with C1RL antibodies and how can they be addressed?

Researchers frequently encounter several challenges when working with C1RL antibodies:

• Background signal in immunostaining:

  • Increase blocking time and concentration

  • Use more stringent washing protocols

  • Consider tissue-specific autofluorescence quenching for IF

• Inconsistent molecular weight detection:

  • C1RL shows variation in apparent molecular weight (calculated 53 kDa vs. observed 70 kDa) due to N-glycosylation

  • Consider deglycosylation treatment to confirm identity

  • Use positive control samples with known C1RL expression

• Tissue-specific optimization requirements:

  • Different fixation protocols may be needed for various tissues

  • Antigen retrieval conditions may require optimization

  • Antibody concentration should be titrated for each application

How does antibody selection impact the quality of C1RL detection in different applications?

Antibody selection significantly influences experimental outcomes. For C1RL studies, consider:

• Polyclonal vs. monoclonal: Current commercial C1RL antibodies are primarily polyclonal , offering broad epitope recognition but potential batch variation

• Species reactivity: Verify cross-reactivity with your model system (human and mouse reactivity confirmed for some antibodies)

• Storage and handling:

  • Store at recommended temperatures (-20°C for many C1RL antibodies)

  • Avoid repeated freeze-thaw cycles

  • Use storage buffers appropriate for the antibody (PBS or glycerol formulations)

• Application-specific validation: An antibody performing well in Western blot may not be optimal for IHC or IF applications

How can computational approaches enhance C1RL antibody design and application?

Recent advances in computational antibody design offer opportunities for improving C1RL-targeted reagents:

Computational methods can predict antibody-antigen interactions, allowing researchers to:

• Design antibodies with improved specificity for C1RL

• Predict cross-reactivity with related proteins

• Optimize antibody stability and expressibility

The AbDesign algorithm represents one such approach, using a three-stage process to optimize both antibody stability and binding energy . Such computational methods can help address challenges in antibody design, particularly for targeting nonideal features like those in C1RL.

For researchers working with existing antibodies, structural modeling of C1RL-antibody interactions can help predict epitope accessibility in different experimental conditions and applications.

What emerging technologies could enhance C1RL antibody applications in research?

Several cutting-edge technologies show promise for advancing C1RL research:

• Single-cell proteogenomics: Integration of antibody-based detection with transcriptomic analysis at single-cell resolution

• Spatial transcriptomics with protein co-detection: Analyzing C1RL protein expression in spatial context alongside gene expression data

• Advanced microscopy techniques:

  • Super-resolution microscopy for detailed subcellular localization

  • Intravital imaging for studying C1RL dynamics in vivo

• Antibody engineering approaches:

  • Nanobodies for improved tissue penetration

  • Bispecific antibodies for functional studies

  • Site-specific conjugation techniques for improved imaging probes

How might C1RL research contribute to immunology and cancer biology?

Current evidence suggests several promising avenues for C1RL research:

• Cancer biomarker development: C1RL upregulation in glioblastoma, hepatocellular carcinoma, and renal cell cancer points to potential diagnostic and prognostic applications

• Immunomodulation: As a complement-related protein, C1RL may influence tumor immune microenvironments

• Therapeutic targeting: Understanding C1RL function could reveal new therapeutic opportunities, particularly in cancers where it serves as a prognostic marker

• Complement system biology: Further characterization of C1RL's role may provide insights into novel complement activation pathways

Future research should focus on validating C1RL's functional significance through carefully designed in vitro and in vivo models, with particular attention to its role in modulating immune responses in disease contexts.