CLCNKB Antibody

What is CLCNKB and why is it important for kidney function research?

CLCNKB (Chloride channel protein ClC-Kb) is a member of the CLC family of voltage-gated chloride channels, which comprises at least 9 mammalian chloride channels. Each channel contains 12 transmembrane domains with intracellular N and C termini. CLCNKB is predominantly expressed in the kidneys, specifically along nephron segments including the thick ascending limb of Henle's loop, convoluted tubule, and collecting duct . The protein is critical for maintaining systemic acid-base and electrolyte homeostasis by mediating chloride conductance in these segments. Additionally, CLCNKB conducts chloride currents in the stria vascularis of the inner ear, establishing the endocochlear potential necessary for normal hearing . Researchers investigating renal physiology, electrolyte disorders, or hearing mechanisms should consider CLCNKB as a key target for investigation.

How do I select the appropriate CLCNKB antibody for my specific application?

When selecting a CLCNKB antibody, consider the following methodological approach:

• Determine your experimental application: Different antibodies are validated for specific applications like Western blot (WB), immunohistochemistry (IHC-P), or immunocytochemistry/immunofluorescence (ICC/IF) .

• Consider species reactivity: Verify that the antibody recognizes CLCNKB in your species of interest. Available antibodies react with human, rat, and/or mouse CLCNKB .

• Antibody type selection:

  • Polyclonal antibodies (like ab236733) offer higher sensitivity but potentially lower specificity

  • Monoclonal antibodies (like EPR8414(2)) provide higher specificity for particular epitopes

• Epitope location: Consider antibodies targeting different regions of CLCNKB. For example, ab236733 targets the C-terminal region (aa 500 to C-terminus), while other antibodies target different domains .

• Cross-reactivity concerns: Due to the 94% sequence identity between CLCNKA and CLCNKB, some antibodies may detect both proteins. For instance, the Anti-CLC-K Antibody (#ACL-004) recognizes both CLC-K1 and CLC-K2 isoforms . If you need isoform specificity, select antibodies validated for CLCNKB selectivity.

What positive controls should I include when validating a new CLCNKB antibody?

For rigorous validation of CLCNKB antibodies, implement these methodological controls:

• Tissue positive controls: Kidney tissue, particularly from thick ascending limb, convoluted tubule, and collecting duct regions where CLCNKB is highly expressed .

• Cell line controls: HEK293T or CHO cells transfected with CLCNKB expression vectors have been successfully used to validate antibody specificity .

• Recombinant protein: Use purified recombinant CLCNKB protein fragments as positive controls for Western blot applications.

• Blocking peptide validation: Include a parallel experiment using the antibody pre-incubated with the immunizing peptide, which should abolish specific signals .

• Knockout/knockdown validation: When available, tissues or cells with CLCNKB knockout/knockdown provide the gold standard for antibody specificity verification.

How can I differentiate between CLCNKA and CLCNKB in my experiments given their high sequence homology?

Distinguishing between these highly homologous proteins (94% sequence identity) requires careful experimental design:

• Isoform-specific antibodies: Select antibodies raised against regions where amino acid sequences differ between CLCNKA and CLCNKB.

• Genetic approaches: Use CLCNKA or CLCNKB knockout models as controls to confirm antibody specificity.

• Expression pattern analysis: CLCNKA and CLCNKB have distinct expression patterns in kidney segments that can help distinguish them:

  • CLCNKA: Predominantly in thin ascending limb of Henle's loop

  • CLCNKB: Expressed in thick ascending limb, distal convoluted tubule, and collecting duct

• RT-PCR with isoform-specific primers: Design primers targeting divergent regions of CLCNKA and CLCNKB mRNAs.

• Electrophysiological properties: In functional studies, CLCNKA and CLCNKB can be differentiated by their distinct electrophysiological properties when expressed with Barttin .

What are the recommended protocols for immunohistochemical detection of CLCNKB in kidney tissue?

For optimal immunohistochemical detection of CLCNKB in kidney tissue:

• Tissue preparation:

  • Fix tissues in 4% paraformaldehyde for 24 hours

  • Process and embed in paraffin

  • Section at 4-6 μm thickness

• Antigen retrieval:

  • Heat-induced epitope retrieval using citrate buffer (pH 6.0) at 95°C for 20 minutes

  • Allow cooling to room temperature for 20 minutes

• Blocking and antibody incubation:

  • Block with 5% normal serum in PBS containing 0.1% Triton X-100 for 1 hour

  • Incubate with primary CLCNKB antibody at 1:200 dilution overnight at 4°C

  • Wash 3 times with PBS

  • Incubate with appropriate secondary antibody for 1 hour at room temperature

• Detection and counterstaining:

  • Develop using DAB or other suitable chromogen

  • Counterstain with hematoxylin

  • Mount with appropriate mounting medium

• Expected pattern: Look for basolateral membrane staining in thick ascending limb, distal convoluted tubule, and collecting duct cells .

What experimental approach should I use to study the interaction between CLCNKB and Barttin?

To investigate CLCNKB-Barttin interactions, employ these methodological approaches:

• Co-immunoprecipitation:

  • Express tagged versions of CLCNKB and Barttin in heterologous expression systems

  • Immunoprecipitate using antibodies against one protein and probe for the other

  • Include appropriate controls (single transfections, unrelated proteins)

• Proximity ligation assay (PLA):

  • Use specific antibodies against CLCNKB and Barttin from different species

  • Apply species-specific PLA probes to detect protein-protein interactions in situ

• FRET/BRET analysis:

  • Generate fusion proteins of CLCNKB and Barttin with appropriate fluorescent/bioluminescent tags

  • Measure energy transfer as an indicator of protein proximity

• Functional co-expression studies:

  • Express CLCNKB with or without Barttin in expression systems

  • Measure chloride currents using patch-clamp electrophysiology

  • Analyze how Barttin affects CLCNKB channel properties like current-voltage relationships and gating

• Trafficking studies:

  • Use fluorescently tagged constructs to monitor localization

  • Compare subcellular distribution of CLCNKB with and without Barttin co-expression

How do different CLCNKB mutations affect antibody binding and experimental results?

CLCNKB mutations can significantly impact antibody binding and experimental outcomes:

• Epitope-specific effects:

  • Mutations within the antibody epitope region may directly prevent antibody binding

  • For example, mutations in the C-terminal region would affect antibodies targeting amino acids 500 to C-terminus

• Expression level alterations:

  • Many mutations cause reduced protein expression or stability

  • Complete gene deletions (common in Bartter syndrome) will eliminate any antibody binding

• Structural conformational changes:

  • Mutations like Gly167Cys can affect protein folding and glycosylation

  • Structurally altered proteins may mask epitopes even if the sequence is intact

• Trafficking alterations:

  • Mutations affecting trafficking (like those disrupting Barttin binding) may alter the subcellular localization pattern in immunostaining experiments

• Protein degradation:

  • Some mutations trigger increased protein degradation, resulting in reduced signal intensity regardless of antibody quality

When working with samples potentially containing CLCNKB mutations, researchers should validate antibody binding using multiple epitope-targeting antibodies and correlate with functional data.

What methodological approaches can detect structural changes in CLCNKB proteins carrying disease-causing mutations?

For detecting structural changes in mutant CLCNKB proteins:

• Glycosylation analysis:

  • Western blot analysis of glycosylation state using PNGase F treatment

  • Compare migration patterns of wild-type and mutant proteins

  • Mutations like Gly167Cys show significantly diminished glycosylation (wild-type: 0.28 ± 0.07 AU vs. Gly167Cys: 0.02 ± 0.01 AU)

• Surface expression quantification:

  • Surface biotinylation assays to quantify membrane-expressed channels

  • Subcellular fractionation to determine protein distribution

  • Immunohistochemical analysis of basolateral membrane localization

• Thermal stability assays:

  • Measure protein stability through thermal shift assays

  • Compare melting temperatures between wild-type and mutant proteins

• Structural analysis techniques:

  • Circular dichroism spectroscopy to assess secondary structure changes

  • Fluorescence spectroscopy to examine tertiary structure alterations

  • Limited proteolysis to identify structurally altered regions

• Functional correlations:

  • Patch-clamp electrophysiology to measure chloride currents

  • Compare current-voltage relationships and gating properties

  • Example data: Gly167Cys mutation shows significantly diminished chloride current compared to wild-type

How can I experimentally distinguish between trafficking defects and functional defects in CLCNKB mutants?

Differentiating between trafficking and functional defects requires a systematic approach:

• Step 1: Surface expression analysis:

  • Surface biotinylation assays quantify channel presence at the membrane

  • Immunofluorescence with membrane markers assesses colocalization

  • Subcellular fractionation separates membrane from intracellular pools

• Step 2: Functional assessment:

  • Patch-clamp electrophysiology measures channel currents

  • Chloride flux assays quantify ion transport activity

  • Compare the ratio of function to surface expression between wild-type and mutants

• Step 3: Rescue experiments:

  • Low-temperature incubation (27°C) rescues many trafficking defects

  • Chemical chaperones (glycerol, DMSO) can enhance surface trafficking

  • Pharmacological potentiators test channel function independently of trafficking

• Step 4: Protein quality control manipulation:

  • Proteasome inhibitors (MG132) block degradation of misfolded proteins

  • Lysosome inhibitors (chloroquine) prevent lysosomal degradation

  • Monitor changes in protein levels and localization

• Step 5: Correlation analysis:

  • Plot function versus surface expression for multiple mutants

  • Trafficking mutants: Normal function when normalized to surface expression

  • Functional mutants: Reduced function despite normal surface expression

For example, the Gly167Cys mutation shows similar surface expression to wild-type CLCNKB in biotinylation and subcellular fractionation experiments but displays significantly reduced chloride currents, indicating a primary functional defect rather than a trafficking problem .

What are the most common problems when using CLCNKB antibodies and how can they be resolved?

How should I optimize Western blot protocols specifically for CLCNKB detection?

For optimal CLCNKB detection by Western blot:

• Sample preparation:

  • Extract proteins using specialized membrane protein buffers containing 1% Triton X-100 or NP-40

  • Include protease inhibitor cocktail to prevent degradation

  • Do not boil samples; instead, heat at 60°C for 5 minutes

  • Use fresh samples whenever possible

• Gel selection and running conditions:

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

  • Load 30-50 μg of total protein per lane

  • Run at lower voltage (80-100V) to improve resolution of membrane proteins

• Transfer optimization:

  • Use wet transfer systems for more efficient transfer of membrane proteins

  • Transfer at 30V overnight at 4°C for improved efficiency

  • Use PVDF membranes (0.45 μm pore size) for better protein retention

• Blocking and antibody incubation:

  • Block with 5% non-fat milk in TBST for 1-2 hours at room temperature

  • Dilute primary antibody 1:200 to 1:1000 in blocking solution

  • Incubate with primary antibody overnight at 4°C

  • Wash extensively (4 × 10 minutes) with TBST

• Detection and interpretation:

  • Expected molecular weight: ~75 kDa (unglycosylated), ~85-90 kDa (glycosylated)

  • Include PNGase F-treated controls to identify glycosylation bands

  • Use kidney tissue lysates as positive controls

What are the critical considerations when designing experiments to study CLCNKB and Barttin interactions?

When investigating CLCNKB-Barttin interactions, consider these critical factors:

• Expression system selection:

  • HEK293T or CHO cells are commonly used and well-validated

  • Oocyte expression systems provide robust functional data

  • Consider polarized epithelial cell lines (MDCK) for trafficking studies

• Construct design:

  • Avoid tags that might interfere with the Barttin-binding interface

  • Consider the position of fluorescent tags for FRET/localization studies

  • Include appropriate linker sequences between the protein and tags

• Expression level control:

  • Use inducible expression systems to control protein levels

  • Co-express CLCNKB and Barttin at physiological ratios

  • Include controls with varying expression levels to assess stoichiometry effects

• Functional readouts:

  • Patch-clamp electrophysiology remains the gold standard

  • Measure both current amplitude and biophysical properties (voltage dependence, kinetics)

  • CLCNKB:BSND channels display mostly linear current-voltage relationships controlled by common gate

• Trafficking assessment:

  • Quantify surface expression relative to total expression

  • Assess basolateral sorting in polarized cells

  • Monitor real-time trafficking using live-cell imaging

How can CLCNKB antibodies be utilized in Bartter syndrome research and diagnostics?

CLCNKB antibodies offer valuable tools for Bartter syndrome research and diagnostics:

• Genotype-phenotype correlation studies:

  • Immunohistochemistry and Western blot analysis to assess CLCNKB expression levels in patient samples

  • Correlation of protein expression with mutation type and clinical severity

  • Analysis of how different mutations affect CLCNKB localization and function

• Pathophysiological mechanisms:

  • Investigation of how CLCNKB deficiency affects downstream signaling pathways

  • Assessment of compensatory mechanisms in kidney tissues

  • Understanding the relationship between CLCNKB dysfunction and symptoms

• Therapeutic development platforms:

  • Screening for compounds that rescue mutant CLCNKB function

  • Testing interventions that enhance surface expression of trafficking-defective mutants

  • Validation of gene therapy approaches targeting CLCNKB

• Diagnostic applications:

  • Development of immunoassays to detect CLCNKB protein levels in accessible samples

  • Creating antibody panels against common mutant forms

  • Combining protein analysis with genetic testing for comprehensive diagnostics

• Model system validation:

  • Verification of CLCNKB knockout/knockin animal models

  • Validation of patient-derived cellular models (iPSCs, organoids)

  • Assessment of tissue-specific expression patterns in experimental models

What novel research directions are emerging from recent CLCNKB structural and functional studies?

Recent advances in CLCNKB research are opening exciting new directions:

• Structure-function relationships:

  • Investigation of how glycosylation affects CLCNKB function, as mutations like Gly167Cys highlight the importance of this post-translational modification

  • Understanding the molecular basis of ion selectivity (chloride > bromide > nitrate > iodide)

  • Characterization of the dual gating mechanism (fast gates controlling each subunit independently and a slow common gate)

• Expanding disease associations:

  • Exploring the relationship between CLCNKB mutations and non-syndromic hearing loss

  • Investigating potential connections between CLCNKB variants and other renal disorders

  • Examining the role of CLCNKB in focal segmental glomerulosclerosis (FSGS)

• Therapeutic innovations:

  • Development of pharmacological chaperones specific for CLCNKB mutants

  • Design of gene therapy approaches targeting the kidney

  • Exploration of small molecule modulators of channel function

• Advanced model systems:

  • Creation of kidney organoids with CLCNKB mutations

  • Development of conditional and tissue-specific knockout models

  • Application of CRISPR-Cas9 for precise genomic editing of CLCNKB

• Regulatory networks:

  • Investigation of transcriptional and post-transcriptional regulation of CLCNKB

  • Exploration of interacting proteins beyond Barttin

  • Understanding the coordinated regulation of CLCNKA and CLCNKB expression

How can researchers effectively study the functional relationship between CLCNKB and hearing loss?

To investigate the CLCNKB-hearing loss connection:

• Inner ear expression analysis:

  • Use immunohistochemistry with CLCNKB antibodies to map expression in cochlear tissues

  • Compare expression patterns between normal and disease models

  • CLCNKB conducts chloride currents in the stria vascularis to establish the endocochlear potential necessary for normal hearing

• Electrophysiological assessments:

  • Measure endocochlear potential in animal models with CLCNKB mutations

  • Conduct patch-clamp studies of strial marginal cells

  • Compare chloride conductance properties between wild-type and mutant tissues

• Combined genotype-phenotype studies:

  • Screen for CLCNKB mutations in cohorts with non-syndromic hearing loss

  • Analyze families with both Bartter syndrome and hearing phenotypes

  • Create comprehensive databases linking specific mutations to audiological profiles

• Functional rescue experiments:

  • Test hearing restoration using gene therapy approaches in animal models

  • Evaluate pharmacological interventions targeting CLCNKB function

  • Assess the efficacy of early intervention strategies

• Developmental studies:

  • Examine the role of CLCNKB during inner ear development

  • Investigate the timeline of CLCNKB expression in relation to cochlear maturation

  • Determine critical periods when CLCNKB function is essential for normal hearing

This approach will provide valuable insights into how CLCNKB dysfunction contributes to hearing impairment and may identify potential therapeutic targets for intervention.