CLCN6 Antibody

What is CLCN6 and why is it significant for neurological research?

CLCN6 is a late endosomal voltage-gated chloride-proton exchanger predominantly expressed in the nervous system. This protein functions as a chloride/proton antiporter and is critical for proper endolysosomal function. Its significance stems from its association with severe neurological diseases, particularly early-onset neurodegenerative disorders. Both loss-of-function and gain-of-function mutations in CLCN6 can cause neuronal pathology, revealing its critical role in maintaining normal neuronal function . Mouse models with disrupted CLCN6 exhibit reduced pain sensitivity and mild cognitive abnormalities resembling mild forms of human neuronal ceroid lipofuscinosis (NCL), further highlighting its importance in neurological research .

What types of CLCN6 antibodies are available for research applications?

CLCN6 antibodies are available in several formats to accommodate different experimental requirements:

These antibodies are validated for various applications including Western Blotting (WB), Enzyme-Linked Immunosorbent Assay (ELISA), Immunocytochemistry/Immunofluorescence (ICC/IF), and Immunohistochemistry (IHC) . They show reactivity with CLCN6 from multiple species including human, mouse, rat, and in some cases, additional species such as monkey, dog, cow, guinea pig, and horse .

How should CLCN6 antibodies be stored and handled to maintain optimal activity?

For optimal preservation of CLCN6 antibody activity, follow these methodological guidelines:

• Store at -20°C for long-term storage (up to one year)

• For frequent use, store at 4°C for up to one month

• Avoid repeated freeze-thaw cycles as they can degrade antibody quality and binding efficiency

• When working with lyophilized forms, reconstitute in sterile water (typically 100 µL) and centrifuge to remove any insoluble material

• Once reconstituted, aliquot the antibody to minimize freeze-thaw cycles if not using immediately

• Most CLCN6 antibodies are supplied in buffers containing preservatives like glycerol (50%) and sometimes sodium azide (0.02%), which help maintain stability

When handling, always wear appropriate personal protective equipment and follow your institution's guidelines for working with biological materials.

What controls should be included when performing experiments with CLCN6 antibodies?

When designing experiments using CLCN6 antibodies, include the following controls to ensure valid and interpretable results:

• Positive control: Use tissues or cell lines known to express CLCN6, primarily neural tissues as CLCN6 is almost exclusively detected in the nervous system

• Negative control: Use tissues from CLCN6 knockout models when available, or tissues known not to express CLCN6

• Primary antibody omission: Process samples without the primary antibody to assess background and non-specific binding of secondary antibodies

• Isotype control: For monoclonal antibodies, include an irrelevant antibody of the same isotype (e.g., IgG2b for clone 2H2)

• Blocking peptide control: When available, pre-incubate the antibody with its specific immunogen peptide to confirm specificity

• Recombinant protein: For certain applications like Western blot, include recombinant CLCN6 protein as a size reference

For Western blotting specifically, GST-tagged recombinant protein fragments can serve as positive controls, with GST tag alone as a negative control to confirm specificity to CLCN6 rather than the tag .

How do mutations in specific structural domains of CLCN6 affect antibody recognition, and what methodological approaches can address these challenges?

Mutations in CLCN6 can alter epitope accessibility or structure, potentially affecting antibody recognition. This is particularly relevant for disease-associated mutations like Y553C and T520A identified in severe neurodegenerative disorders . To address these challenges:

• Epitope mapping approach: Use antibodies targeting different regions of CLCN6 (N-terminal, C-terminal, internal regions) to determine if specific mutations affect recognition at particular epitopes

• Competitive binding assays: Employ competitive ELISAs with wild-type and mutant peptides to quantify affinity changes

• Cross-validation methodology:

  • Compare results from multiple antibodies targeting different epitopes

  • Confirm protein detection using orthogonal methods (mass spectrometry)

  • Validate with genetic approaches (siRNA knockdown, CRISPR-edited cells)

• Structural analysis integration: Correlate antibody binding efficacy with the known structural changes induced by specific mutations, particularly in relation to the Y553/F317/T520 interaction network identified as a hotspot for disease-causing mutations

For mutations in the ATP-binding regions (R833, H851, H630), researchers should consider how these might affect protein conformation and consequently antibody recognition, especially when studying functional aspects of CLCN6 .

What are the critical considerations when developing immunohistochemistry protocols for CLCN6 detection in neural tissues?

Developing effective immunohistochemistry protocols for CLCN6 in neural tissues requires addressing several technical challenges:

• Antigen retrieval optimization:

  • Compare heat-induced epitope retrieval methods (citrate buffer pH 6.0 vs. EDTA buffer pH 9.0)

  • Evaluate enzymatic retrieval approaches for preserved membrane protein structure

  • Optimize retrieval duration based on fixation conditions (10-30 minutes)

• Signal amplification strategies:

  • For low abundance detection, implement tyramide signal amplification

  • Consider biotin-free detection systems to reduce background in neural tissues

  • Dilution ranges of 1:500-1:1000 are recommended for IHC applications with certain antibodies

• Co-localization studies:

  • Pair CLCN6 detection with endolysosomal markers (LAMP1) to confirm proper localization

  • Use markers for axon initial segments where ClC-6 knockout mice show pathological changes

  • Include sequential staining protocols to avoid cross-reactivity

• Tissue preparation considerations:

  • Fresh frozen versus fixed tissue comparisons (perfusion fixation typically preserves CLCN6 epitopes better)

  • Section thickness optimization (typically 5-10μm for good resolution)

  • Post-fixation protocols to maintain membrane integrity

• Specificity verification:

  • Include tissues from ClC-6 knockout mice as definitive negative controls

  • Perform absorption controls with immunizing peptides when available

The validation should include multiple neural regions, particularly those showing pathology in CLCN6-associated neurodegeneration models.

How can researchers distinguish between wild-type CLCN6 and disease-associated mutant forms in experimental systems?

Distinguishing between wild-type CLCN6 and disease-associated mutant forms requires multiple complementary approaches:

• Antibody-based differentiation strategies:

  • Develop mutation-specific antibodies targeting known disease mutations (Y553C, T520A)

  • Use proximity ligation assays to detect specific protein-protein interactions altered by mutations

  • Employ native PAGE conditions that might separate conformationally distinct forms

• Functional readouts:

  • Measure chloride transport activity differences between wild-type and mutant forms

  • Assess ATP binding capacity and its effect on ion transport in different mutants

  • Quantify formation of LAMP1-positive giant vacuoles characteristic of Y553C mutation

• Cellular localization analysis:

  • Compare subcellular distribution patterns using high-resolution microscopy

  • Measure co-localization coefficients with endolysosomal markers

  • Assess trafficking dynamics using pulse-chase experimental designs

• Structural biology approaches:

  • Use structural information from cryo-EM studies to predict and verify conformational differences

  • Analyze voltage-dependent gating differences between wild-type and mutant proteins

  • Examine the integrity of the Y553/F317/T520 interaction network in different mutants

• Expression system considerations:

  • Compare heterologous expression systems (HEK293) with neuronal cultures for accurate representation of phenotypes

  • Develop knockin cell lines expressing disease-associated mutations at endogenous levels

These approaches should be tailored to the specific mutation being studied, as gain-of-function and loss-of-function mutations will require different experimental strategies for detection and characterization.

What methodological approaches can resolve conflicting data regarding CLCN6 expression patterns across different tissues?

When addressing conflicting reports about CLCN6 expression patterns, consider implementing these methodological strategies:

• Comprehensive antibody validation workflow:

  • Test multiple antibodies targeting different CLCN6 epitopes

  • Validate each antibody using knockout tissues/cells as negative controls

  • Perform peptide competition assays to confirm specificity

  • Compare protein detection with transcript levels (qPCR, RNA-seq)

• Species-specific consideration framework:

  • Early studies indicated broad transcription of CLCN6, but protein detection showed expression nearly exclusively in the nervous system

  • Systematically compare expression across species using consistent detection methods

  • Account for developmental stage-specific expression patterns

• Technique triangulation approach:

  Technique	Advantages	Limitations	Implementation Notes
  Western blot	Quantitative, size verification	Poor spatial information	Include positive and negative tissues confirmed by other methods
  IHC/IF	Spatial localization	Fixation artifacts	Use multiple fixation protocols
  In situ hybridization	mRNA detection	Post-transcriptional regulation not detected	Compare with protein detection methods
  Single-cell transcriptomics	Cell-type resolution	Protein levels may differ	Validate with proteomics

• Methodological controls for transcript-protein discrepancies:

  • Investigate post-transcriptional regulation mechanisms

  • Assess protein stability and turnover rates in different tissues

  • Examine alternative splicing that might affect epitope availability

• Experimental design considerations:

  • Standardize tissue harvesting procedures

  • Control for age, sex, and strain variables in animal models

  • Use quantitative approaches (quantitative Western blotting, flow cytometry) when possible

By implementing these approaches systematically, researchers can develop a more accurate understanding of CLCN6 expression patterns and resolve apparent discrepancies in the literature.

What are the optimal methodological approaches for studying the interaction between CLCN6 and ATP, and how can antibodies facilitate this research?

To investigate the newly recognized ATP-binding properties of CLCN6 and the enhancement of ion transport activity , researchers should consider these methodological approaches:

• Structural binding site analysis:

  • Use site-directed mutagenesis targeting key residues in ATP binding (R833, H851, H630)

  • Implement proximity-based labeling techniques (crosslinking, photoaffinity labeling)

  • Develop antibodies specifically recognizing the ATP-bound conformation

• Functional transport assays:

  • Utilize patch-clamp electrophysiology with controlled ATP concentrations

  • Employ chloride-sensitive fluorescent indicators in live cell imaging

  • Compare wild-type and ATP binding site mutants in parallel experiments

• ATP-binding characterization:

  • Perform isothermal titration calorimetry (ITC) to measure binding kinetics

  • Use microscale thermophoresis for quantitative binding analysis

  • Implement ATP competition assays with structural analogs

• Antibody-facilitated approaches:

  • Develop conformation-specific antibodies that preferentially recognize ATP-bound states

  • Use antibodies in pull-down assays coupled with ATP detection methods

  • Apply antibodies in proximity ligation assays to detect CLCN6-ATP interactions

• Experimental design considerations:

  • Include internal controls for ATP specificity (GTP, ADP testing)

  • Control intracellular ATP levels systematically (metabolic inhibitors, permeabilization)

  • Account for physiological ATP concentration ranges (1-10 mM)

The combination of these approaches will provide comprehensive insights into the physiological significance of ATP binding to CLCN6 and its potential implications in disease states, particularly in the context of neurodegeneration.

How can researchers effectively study the relationship between CLCN6 dysfunction and lysosomal storage disorders using antibody-based techniques?

To investigate the connection between CLCN6 dysfunction and lysosomal storage disorders (particularly forms of neuronal ceroid lipofuscinosis), implement these methodological approaches:

• Cellular phenotype characterization:

  • Quantify LAMP1-positive vesicle formation in cells expressing disease-associated mutants

  • Implement co-localization analysis between CLCN6 and lysosomal markers

  • Measure vesicular pH using ratiometric probes in wild-type versus mutant cells

• Storage material analysis:

  • Combine CLCN6 immunolabeling with detection of storage materials (autofluorescent lipopigments)

  • Use electron microscopy with immunogold labeling to examine ultrastructural changes

  • Apply dual immunofluorescence with antibodies against saposin D and subcocllin to characterize storage material components

• Axon initial segment pathology assessment:

  • Develop protocols for co-detection of CLCN6 and axon initial segment markers

  • Quantify enlargement of axon initial segments in neuronal cultures expressing mutant CLCN6

  • Compare findings with those from ClC-6 knockout mice and human NCL samples

• Comparative models approach:

  Model System	Advantages	Key Readouts	Antibody Applications
  CLCN6 knockout mice	In vivo physiological context	Storage material accumulation, behavioral phenotypes	IHC, WB for compensatory changes
  Patient-derived cells	Human disease relevance	Lysosomal morphology, function	Live-cell imaging with immunolabeling
  CRISPR-edited neurons	Precise genetic modification	Electrophysiology, trafficking	Super-resolution microscopy

• Therapeutic intervention assessment:

  • Measure CLCN6 levels and localization during treatment trials

  • Monitor clearance of storage material using quantitative immunofluorescence

  • Track changes in endolysosomal function with activity-based probes

These approaches collectively enable researchers to establish mechanistic links between CLCN6 dysfunction and the pathogenesis of lysosomal storage disorders, potentially identifying new therapeutic targets for neurodegenerative conditions.