CLC-A Antibody

What is CLC and what biological functions does it serve?

Cardiotrophin-like cytokine (CLC), also known as novel neurotrophin-1 (NNT-1) and B cell stimulating factor (BSF-3), is a 22-25 kDa member of the IL-6 family of cytokines. Human CLC is synthesized as a 225 amino acid precursor containing a 27 amino acid signal sequence and a 198 amino acid mature region . Despite having a signal sequence, CLC is not secreted unless it forms a non-covalent dimer with either CLF (cytokine-like factor) or soluble CNTFR alpha (ciliary neurotrophic factor receptor alpha) .

CLC serves multiple biological functions, including:

• Acting as a trophic factor for motor neurons

• Stimulating ACTH release from corticotrophs

• Inducing IgE synthesis

• Promoting B cell proliferation

CLC is expressed in embryonic muscle, lung epithelium, and various mesenchymal regions during development .

How does CLC signaling work in cellular systems?

CLC signals through a tripartite receptor complex composed of gp130, LIFR beta (leukemia inhibitory factor receptor beta), and CNTFR alpha (ciliary neurotrophic factor receptor alpha) . This signaling mechanism is characteristic of the IL-6 family of cytokines, which typically utilize gp130 as a common signal-transducing receptor component.

The signaling process follows these steps:

• CLC first dimerizes with either CLF or soluble CNTFR alpha to enable secretion

• The CLC-containing complex binds to membrane-bound CNTFR alpha

• This interaction facilitates recruitment of gp130 and LIFR beta

• The completed receptor complex activates downstream signaling cascades, including JAK-STAT, MAPK, and PI3K pathways

Within the IL-6 family, human CLC shares approximately 29% amino acid sequence identity with cardiotrophin-1, indicating both shared and distinct functions .

What is the difference between CLC antibodies and ClC-a chloride channel antibodies?

This is an important distinction for researchers to understand. While the abbreviations appear similar, they represent entirely different biological molecules:

In Drosophila research, ClC-a refers specifically to a chloride channel whose mammalian ortholog CLCN2 is expressed in glial cells. Defective function of this channel in humans results in leukodystrophies accompanied by cognitive impairment .

How can CLC antibodies be used to investigate neuronal development and neuropathology?

CLC antibodies serve as valuable tools for investigating neuronal development and pathology due to CLC's role as a neurotrophic factor. Methodological approaches include:

• Immunohistochemistry and immunofluorescence: These techniques allow visualization of CLC expression patterns in developing neural tissues and in neuropathological conditions. Use perfusion-fixed tissue sections (10-20 μm) with appropriate antigen retrieval methods for optimal staining.

• Neutralization studies: CLC antibodies can block the trophic effects of CLC on motor neurons in vitro. This approach requires:

  • Primary motor neuron cultures from embryonic spinal cord

  • Addition of CLC antibodies (typically 0.1-10 μg/mL) to neutralize endogenous or exogenous CLC

  • Quantification of neuronal survival, neurite outgrowth, or molecular markers

• In vivo functional studies: Intracerebroventricular injection of CLC antibodies can help determine the role of endogenous CLC in neural development or regeneration after injury. These studies should include appropriate controls and dose-response assessments.

The specificity of the antibody is critical for these applications, and validation should include Western blotting against recombinant CLC and tissue lysates, along with peptide competition assays to confirm binding specificity.

What considerations should be made when using CLC antibodies to study its interactions with receptor complexes?

When investigating CLC interactions with its receptor complex (gp130/LIFR beta/CNTFR alpha), researchers should consider:

• Co-immunoprecipitation protocols: Use gentle lysis buffers (containing 1% NP-40 or 0.5% Triton X-100) to preserve protein-protein interactions. Pre-clear lysates thoroughly to reduce non-specific binding.

• Sequential immunoprecipitation: This approach can help identify multiprotein complexes:

  • First immunoprecipitate with anti-CLC antibody

  • Elute under mild conditions

  • Perform second immunoprecipitation with antibodies against receptor components

• Proximity ligation assays: This technique can detect protein-protein interactions in situ with high sensitivity and specificity, requiring two primary antibodies from different species targeting CLC and its receptor components.

• Biolayer interferometry: As demonstrated in research on similar cytokines, this technique can measure binding kinetics between CLC and its receptors in real-time. Consider using a setup similar to what has been described for other cytokines: "For simultaneous binding of multiple antigens to one antibody, the mAb of interest (10 μg/mL) was loaded onto AHC biosensors until a layer thickness of 1 nm was reached" .

These methods require careful antibody selection, as the epitope recognized by the CLC antibody should not interfere with the receptor binding sites unless this interference is part of the experimental design.

How do CLC antibodies compare to other cytokine detection systems in sensitivity and specificity?

When comparing CLC antibodies to other cytokine detection systems, consider these methodological insights:

For optimal specificity when using CLC antibodies, researchers should:

• Validate antibodies against recombinant human CLC protein (Leu28-Phe225)

• Include appropriate blocking steps to minimize non-specific binding

• Use antibodies raised against species-specific CLC epitopes when conducting cross-species research (human to mouse CLC is 96% identical at the amino acid level)

What are the optimal storage and handling conditions for CLC antibodies?

Proper storage and handling of CLC antibodies is critical for maintaining antibody function and experimental reproducibility. Based on established protocols for similar antibodies, follow these guidelines:

• Storage conditions:

  • Long-term storage: -20°C to -70°C for up to 12 months from date of receipt

  • Medium-term storage (reconstituted): 2-8°C under sterile conditions for up to 1 month

  • Extended storage (reconstituted): -20°C to -70°C under sterile conditions for up to 6 months

• Handling precautions:

  • Use a manual defrost freezer and avoid repeated freeze-thaw cycles

  • Aliquot reconstituted antibody to minimize freeze-thaw cycles

  • Centrifuge vials briefly before opening to collect all liquid

  • When diluting, use sterile buffers containing carrier protein (0.1-1% BSA or serum)

• Working dilutions:

  • Determine optimal dilutions empirically for each application

  • Document lot-to-lot variation with standard samples

  • Prepare fresh working dilutions on the day of experiment when possible

These recommendations are derived from standard practices for research antibodies similar to the Human CLC Antibody (AF962) described in the literature .

What controls should be included when using CLC antibodies in Western blotting and immunoprecipitation?

When using CLC antibodies for Western blotting and immunoprecipitation, include these essential controls:

• Western blotting controls:

  • Positive control: Recombinant human CLC protein (Leu28-Phe225)

  • Negative control: Lysate from cells known not to express CLC

  • Loading control: Probing for housekeeping proteins (β-actin, GAPDH)

  • Antibody specificity control: Pre-absorption with recombinant CLC

  • Secondary antibody control: Omitting primary antibody

• Immunoprecipitation controls:

  • Input sample: 5-10% of pre-IP lysate

  • Isotype control: Irrelevant antibody of same isotype and concentration

  • Bead-only control: Protein A/G beads without antibody

  • Reciprocal IP: When studying interactions, perform IPs with antibodies against presumed interacting partners

• Additional validation approaches:

  • siRNA/shRNA knockdown: Reduced signal supports antibody specificity

  • Overexpression: Increased signal in transfected cells confirms specificity

  • Cross-linking: Consider using DSS or formaldehyde (0.5-2%) to stabilize weak or transient interactions

For optimal results, pre-clear lysates thoroughly and use IP lysis buffers that maintain protein-protein interactions if co-IP of receptor complexes is intended.

How should CLC antibodies be validated for immunohistochemistry applications?

Thorough validation of CLC antibodies for immunohistochemistry (IHC) requires a methodical approach:

• Initial validation steps:

  • Positive control tissues: Based on known expression patterns, include embryonic muscle, lung epithelium, and areas with mesenchymal tissues

  • Negative control tissues: Tissues known not to express CLC

  • Blocking peptide control: Pre-incubation of antibody with excess antigen should abolish specific staining

  • Secondary antibody control: Omit primary antibody to assess background

• Optimization parameters:

  • Fixation: Compare paraformaldehyde (4%), formalin, and other fixatives

  • Antigen retrieval: Test heat-induced (citrate buffer pH 6.0, EDTA pH 8.0) and enzymatic methods

  • Antibody concentration: Perform titration series (0.1-10 μg/mL)

  • Incubation conditions: Compare overnight at 4°C vs. shorter incubations at room temperature

• Correlation with other methods:

  • In situ hybridization: Compare protein localization with mRNA expression

  • Fluorescent reporter models: If available, compare with GFP-tagged CLC expression

  • Western blot: Confirm antibody detects a band of appropriate molecular weight in the same tissues

Document all validation steps with images and quantitative assessments to ensure reproducibility across different experimental conditions and tissue samples.

How can researchers address potential cross-reactivity issues with CLC antibodies?

Cross-reactivity is a common challenge when working with antibodies against IL-6 family cytokines due to structural similarities. To address this issue:

• Cross-reactivity assessment protocol:

  • Perform Western blots against recombinant proteins from related IL-6 family members (CT-1, CNTF, LIF)

  • Conduct competitive ELISAs with related cytokines as competitors

  • Use cells expressing individual IL-6 family members for immunocytochemistry comparisons

• Data interpretation guidelines:

  • Establish signal intensity ratios between target and potential cross-reactants

  • Consider a threshold (e.g., <10% cross-reactivity) for acceptable specificity

  • Document any confirmed cross-reactivity in your experimental reports

• Mitigation strategies:

  • Pre-absorb antibodies with related proteins before use

  • Use multiple antibodies targeting different epitopes of CLC

  • Confirm findings with complementary techniques (e.g., mass spectrometry)

  • Consider genetic approaches (knockout/knockdown) to validate antibody specificity

When interpreting results, be particularly cautious about potential cross-reactivity with cardiotrophin-1, which shares approximately 29% amino acid sequence identity with CLC .

What factors can affect the reproducibility of experiments using CLC antibodies?

Several factors can influence experimental reproducibility when using CLC antibodies:

• Antibody-related factors:

  • Lot-to-lot variation: Different production batches may vary in affinity/specificity

  • Antibody degradation: Improper storage or handling can reduce activity

  • Concentration inconsistencies: Errors in dilution or evaporation during storage

• Sample preparation variables:

  • Fixation protocols: Overfixation can mask epitopes

  • Protein extraction methods: Different lysis buffers yield different protein conformations

  • Post-translational modifications: Glycosylation state of CLC may affect antibody recognition

• Experimental conditions:

  • Temperature fluctuations during incubation steps

  • Buffer composition changes between experiments

  • Blocking reagent effectiveness and consistency

• Methodological approach to improve reproducibility:

  • Maintain detailed records of antibody lots, dilutions, and protocols

  • Include standard samples across experiments for normalization

  • Establish quantitative acceptance criteria before beginning experiments

  • Perform technical replicates and biological replicates to assess variability

A systematic approach to documenting these variables is essential for troubleshooting inconsistent results and ensuring experimental reproducibility.

How can researchers distinguish between conflicting data obtained with different CLC antibodies?

When faced with conflicting results from different CLC antibodies, employ this methodological framework:

• Epitope mapping and comparison:

  • Determine which regions of CLC each antibody recognizes

  • Assess if antibodies detect different isoforms or post-translationally modified variants

  • Consider if antibody binding sites overlap with functional domains of CLC

• Validation hierarchy approach:

  • Prioritize antibodies validated with knockout/knockdown controls

  • Compare results with orthogonal methods not relying on antibodies

  • Test antibodies on recombinant CLC with known modifications or mutations

• Systematic resolution protocol:

  • Use multiple antibodies simultaneously in the same experiment

  • Employ antibody cocktails to increase detection sensitivity

  • Conduct sequential probing with different antibodies on the same samples

  • Perform immunodepletion experiments to identify exclusive vs. overlapping populations

• Analytical framework for data integration:

  • Develop a weighted scoring system based on validation strength

  • Consider biological context when interpreting conflicting results

  • Report discrepancies transparently in publications rather than selecting only "favorable" results

Remember that discrepancies between antibodies may reveal important biological insights about different forms or conformations of CLC in various contexts.

How might CLC antibodies be used to investigate the role of CLC in neurological disorders?

CLC antibodies offer promising approaches for investigating neurological disorders:

• Methodological framework for disease models:

  • Utilize CLC antibodies for comparative immunohistochemistry in animal models of motor neuron diseases, comparing expression patterns with control tissues

  • Develop tissue microarrays of human neurological disease samples for high-throughput screening of CLC expression

  • Combine with cell-type specific markers to identify changes in CLC-responsive populations

• Functional studies in disease contexts:

  • Apply CLC neutralizing antibodies to disease model systems to determine if blocking CLC signaling modifies disease progression

  • Use non-neutralizing antibodies to track CLC distribution during disease development

  • Implement conditional knockout models with antibody validation to determine cell-specific contributions

• Translational applications:

  • Develop protocols for CLC detection in cerebrospinal fluid as potential biomarkers

  • Investigate CLC and its receptor components in post-mortem tissues from patients with motor neuron diseases

  • Correlate CLC levels with disease severity or progression using quantitative immunoassays

This research direction is particularly relevant given CLC's role as a neurotrophic factor and the known connection between chloride channels (which share the CLC abbreviation but are distinct proteins) and leukodystrophies with cognitive impairment .

What emerging technologies might enhance the utility of CLC antibodies in research?

Emerging technologies are expanding the research applications of antibodies, including those targeting CLC:

• Advanced imaging approaches:

  • Super-resolution microscopy for nanoscale localization of CLC and its receptors

  • Expansion microscopy to physically enlarge specimens for improved visualization

  • Light-sheet microscopy for rapid 3D imaging of CLC distribution in intact tissues

  • Intravital microscopy for tracking CLC-expressing cells in living organisms

• Single-cell methodologies:

  • CyTOF (mass cytometry) for highly multiplexed detection of CLC alongside dozens of other markers

  • Spatial transcriptomics combined with CLC immunostaining to correlate protein expression with transcriptional profiles

  • Microfluidic antibody-based capture systems for isolating CLC-secreting cells

• Antibody engineering applications:

  • Bispecific antibodies targeting CLC and its receptor components simultaneously, similar to the trispecific checkpoint inhibitor concept described in the literature

  • Antibody fragments with improved tissue penetration for in vivo imaging

  • Antibody-drug conjugates for targeting cells expressing CLC receptors

These technological approaches extend beyond traditional applications and open new possibilities for understanding CLC biology in complex systems.

How can bioinformatics approaches enhance CLC antibody research and validation?

Bioinformatics tools can significantly improve CLC antibody research:

• Epitope prediction and antibody design:

  • Computational prediction of immunogenic epitopes specific to CLC but not related IL-6 family members

  • Molecular dynamics simulations to predict antibody-antigen interactions

  • Virtual screening of antibody libraries against modeled CLC structures

  • In silico affinity maturation to design improved CLC antibodies

• Cross-reactivity analysis framework:

  • Sequence alignment tools to identify regions of homology between CLC and related proteins

  • Structural comparison algorithms to predict potential cross-reactivity based on 3D epitope similarity

  • Machine learning approaches to predict antibody specificity from sequence and structural features

• Integrated data analysis workflows:

  • Automated image analysis pipelines for quantitative immunohistochemistry

  • Statistical frameworks for comparing antibody performance across multiple validation methods

  • Data visualization tools for integrating antibody binding data with functional outcomes

These computational approaches can help researchers select the most appropriate antibodies for specific applications and interpret complex datasets generated using CLC antibodies.