CLCNKA Antibody

What is CLCNKA and why is it significant in physiological research?

CLCNKA (chloride channel Ka) is a critical membrane protein with a calculated molecular weight of 75 kDa, though it typically presents at approximately 70 kDa in experimental conditions . This channel protein primarily functions in chloride transport across cell membranes and plays significant roles in renal and auditory physiology. The protein is encoded by the CLCNKA gene (NCBI Gene ID: 1187) and is particularly important in kidney function, which explains its abundant expression in renal tissue. Research into CLCNKA provides insights into electrolyte homeostasis, blood pressure regulation, and associated channelopathies. Antibodies targeting CLCNKA are essential tools for investigating its expression, localization, and functional alterations in both normal physiology and disease states.

How should researchers select the appropriate CLCNKA antibody for their specific application?

Selecting the optimal CLCNKA antibody requires consideration of multiple experimental factors. First, determine the target region of interest - whether N-terminal (AA 20-47), C-terminal, or other specific amino acid sequences (e.g., AA 470-644, AA 567-616, AA 626-671) . Each region may provide different accessibility depending on your experimental conditions. Second, assess species reactivity requirements - available antibodies demonstrate reactivity with human, mouse, and rat samples, with some showing predicted reactivity to pig, bovine, and dog samples . Third, consider the required applications - while most CLCNKA antibodies work for Western blotting and ELISA, only select products are validated for immunohistochemistry, immunoprecipitation, or immunofluorescence . Finally, evaluate clonality - most commercial CLCNKA antibodies are polyclonal rabbit IgG, which offers good sensitivity but may have batch-to-batch variation .

What are the established dilution ranges for CLCNKA antibodies across different experimental applications?

CLCNKA antibodies require specific dilution ranges for optimal results depending on the application:

These ranges provide starting points, but optimal dilutions should be determined empirically for each specific experimental system and antibody lot . For Western blotting applications, some antibodies perform best in the middle range (1:1000-1:2000), while others may require less dilution for optimal signal detection . Buffer composition, incubation time, and temperature also influence antibody performance beyond simple dilution factors.

What sample preparation protocols optimize CLCNKA detection in Western blotting?

For effective CLCNKA detection by Western blotting, sample preparation should address protein extraction, denaturation, and loading considerations. Begin with fresh or properly preserved tissues/cells - human kidney tissue, mouse kidney/pancreas tissue, and HEK-293 cells have demonstrated positive CLCNKA expression in validation studies . Use a lysis buffer containing protease inhibitors to prevent degradation of the target protein. Since CLCNKA is a membrane protein, consider using detergent-enriched buffers (e.g., RIPA with 1% NP-40 or Triton X-100) to enhance solubilization.

How should researchers design proper controls for experiments using CLCNKA antibodies?

Designing rigorous controls for CLCNKA antibody experiments is essential for result validation. Primary controls should include:

• Positive tissue/cell controls: Human kidney tissue, HEK-293 cells, mouse kidney, and mouse pancreas tissue have demonstrated reliable CLCNKA expression .

• Negative controls: Tissues known to have minimal CLCNKA expression, or cell lines where CLCNKA has been knocked down/out via siRNA or CRISPR techniques.

• Antibody controls:

  • Primary antibody omission control - to assess secondary antibody specificity

  • Isotype control - rabbit IgG at matching concentration to assess non-specific binding

  • Blocking peptide competition - pre-incubating the antibody with the immunizing peptide should dramatically reduce specific signal

• Loading/processing controls: For Western blotting, include housekeeping proteins (β-actin, GAPDH); for IHC, use sequential tissue sections with established markers for tissue components.

Particularly for advanced applications, consider using CLCNKA-overexpression systems as positive controls and CLCNKA-knockout/knockdown systems as negative controls to definitively establish antibody specificity.

What are the critical parameters for successful immunohistochemistry using CLCNKA antibodies?

Successful immunohistochemistry (IHC) with CLCNKA antibodies depends on several critical parameters. Antigen retrieval is particularly important - the recommended protocol specifies TE buffer at pH 9.0, though citrate buffer at pH 6.0 may serve as an alternative . This step is crucial for unmasking epitopes that may be obscured during fixation. Tissue fixation should be optimized; overfixation can reduce antibody accessibility while underfixation may compromise tissue morphology.

Blocking procedures should include both protein blocking (typically 5-10% normal serum) and peroxidase blocking if using HRP-based detection systems. The recommended antibody dilution range for IHC is 1:400-1:1000 , but pilot experiments with a dilution series may be necessary for each new tissue type or lot of antibody. Incubation conditions (time, temperature) significantly impact staining quality - overnight incubation at 4°C often yields optimal signal-to-noise ratio for CLCNKA detection.

Post-primary antibody washing steps should be thorough to remove unbound antibody and reduce background. For visualization, both DAB and AEC chromogens have been successfully used with CLCNKA antibodies, though DAB offers greater stability for long-term storage. Multiple tissue types have been validated for CLCNKA IHC, including human kidney, placenta, testis, skin, spleen, lung, and ovary tissues , providing options for positive controls.

Why might there be discrepancies between calculated (75 kDa) and observed (70 kDa) molecular weights for CLCNKA in Western blotting?

The discrepancy between calculated (75 kDa) and observed (70 kDa) molecular weights for CLCNKA in Western blotting can be attributed to several factors that researchers should consider when interpreting their results. Post-translational modifications can significantly impact protein migration patterns - differential glycosylation states may reduce apparent molecular weight, while phosphorylation can increase or decrease mobility depending on the protein's structure. Alternative splicing of CLCNKA may generate isoforms with different molecular weights than the canonical form.

Protein folding characteristics, even under denaturing conditions, can affect migration rates - highly hydrophobic membrane proteins like CLCNKA often bind more SDS and migrate faster than predicted by their amino acid sequence alone. Experimental conditions such as gel percentage, buffer system, and running conditions also impact relative migration rates. Additionally, proteolytic processing during sample preparation may generate fragments of the full-length protein.

To distinguish between these possibilities, researchers can compare samples prepared under different conditions (varying detergents, denaturants, or reducing agents) or use additional antibodies targeting different epitopes to confirm the identity of the observed band. Mass spectrometry analysis of the isolated protein band would provide definitive information about its identity and any modifications.

What strategies can address weak or non-detectable signals when using CLCNKA antibodies?

When encountering weak or absent signals with CLCNKA antibodies, implement a systematic troubleshooting approach. First, verify protein expression in your samples - CLCNKA has demonstrated reliable expression in kidney tissues, but expression may be limited in other tissues . Consider enriching for membrane fractions since CLCNKA is a membrane protein and may be diluted in whole-cell lysates.

Optimize antibody concentration - try a dilution series extending beyond the recommended range (e.g., 1:500-1:4000 for WB) . Increase antibody incubation time or temperature, such as overnight at 4°C for Western blotting. Enhance detection sensitivity by using amplification systems like biotin-streptavidin or tyramide signal amplification for very low abundance targets.

Modify blocking conditions - excessive blocking can mask epitopes; try different blocking agents (BSA vs. non-fat milk) or reduce blocking duration. For Western blotting, ensure efficient protein transfer by using lower voltage transfers for longer durations, which benefits larger proteins like CLCNKA. Consider using different membrane types (PVDF may bind more protein than nitrocellulose) and try reducing stringency of wash steps (lower salt concentration or reduced detergent in wash buffers).

If these approaches fail, the epitope may be masked or altered in your specific application. Try a different CLCNKA antibody targeting an alternative region of the protein .

How can researchers reduce background and non-specific binding when using CLCNKA antibodies?

High background and non-specific binding can significantly compromise CLCNKA antibody experiments. To address these issues, optimize blocking procedures by testing different blocking agents (5% BSA, 5% non-fat milk, commercial blocking solutions) and extending blocking time to 1-2 hours at room temperature. Increase the number and duration of wash steps between antibody incubations, using TBS-T or PBS-T with 0.1-0.3% Tween-20 for Western blotting, or 0.05-0.1% for IHC applications.

For Western blotting specifically, pre-adsorb secondary antibodies with tissue extracts from the species being studied to remove cross-reactive antibodies. Use higher dilutions of both primary (within the 1:1000-1:4000 range for CLCNKA) and secondary antibodies, as excessive antibody concentrations often contribute to background. Add 0.1-0.5% non-ionic detergent (Triton X-100) to antibody dilution buffers to reduce non-specific hydrophobic interactions.

For immunohistochemistry, include an avidin/biotin blocking step when using biotin-based detection systems, as endogenous biotin in tissues can cause high background. Consider antigen retrieval optimization - while TE buffer at pH 9.0 is recommended for CLCNKA antibodies, over-retrieval can expose non-specific binding sites . If background persists, try titrating primary antibody exposure time and include additional blocking with 5-10% serum from the species in which the secondary antibody was raised.

How can CLCNKA antibodies be optimized for co-immunoprecipitation studies investigating protein-protein interactions?

Co-immunoprecipitation (co-IP) with CLCNKA antibodies requires specific optimization to preserve native protein complexes. Begin with mild lysis conditions - use buffers containing 0.5-1% NP-40 or 0.5-1% Digitonin rather than stronger detergents like SDS that disrupt protein-protein interactions. Add phosphatase and protease inhibitor cocktails to preserve both target proteins and their modification states. Perform all steps at 4°C to minimize complex dissociation.

For CLCNKA antibody selection, choose antibodies specifically validated for immunoprecipitation applications , as not all antibodies that work in Western blotting will efficiently immunoprecipitate their targets. The recommended amount is 0.5-4.0 μg of antibody per 1.0-3.0 mg of total protein lysate . Pre-clear lysates with Protein A/G beads before adding the CLCNKA antibody to reduce non-specific binding.

For binding, incubate antibody with lysate for 2-4 hours or overnight at 4°C with gentle rotation, followed by addition of Protein A/G beads for 1-2 hours. Wash conditions are critical - use at least 4-5 washes with decreasing salt concentrations to maintain specificity while preserving interactions. Elute protein complexes using either low pH glycine buffer (pH 2.5-3.0) followed by immediate neutralization, or by boiling in sample buffer.

For detection of interaction partners, perform Western blot analysis with antibodies targeting suspected interacting proteins. Consider reverse co-IP experiments (using antibodies against predicted interaction partners to pull down CLCNKA) to validate interactions from both directions.

What are the most effective tissue preparation protocols for studying CLCNKA localization across different organ systems?

CLCNKA localization studies require tissue-specific optimization across different organ systems. For renal tissue, where CLCNKA is highly expressed, perfusion fixation with 4% paraformaldehyde provides superior preservation of membrane proteins compared to immersion fixation . For other validated tissues (placenta, testis, skin, spleen, lung, ovary) , immersion fixation in 10% neutral buffered formalin for 24-48 hours is generally effective.

Cryosectioning offers advantages for preserving antigenicity - fix tissues briefly (10-30 minutes) in 4% paraformaldehyde, cryoprotect in 30% sucrose, embed in OCT compound, and section at 8-12 μm thickness. For paraffin embedding, limit fixation time (24 hours maximum) and process tissues carefully to prevent over-dehydration. Cut sections at 4-6 μm thickness for optimal antibody penetration.

Antigen retrieval methods significantly impact CLCNKA detection - TE buffer at pH 9.0 is recommended as the primary method, with citrate buffer at pH 6.0 as an alternative . For multi-color immunofluorescence studies detecting CLCNKA alongside other markers, perform sequential antigen retrieval if co-staining antibodies have different retrieval requirements.

Tissue permeabilization should be gentle for membrane proteins - use 0.1-0.3% Triton X-100 or 0.1% saponin in PBS for 10-15 minutes. For immunofluorescence applications, include Sudan Black B treatment (0.1-0.3% in 70% ethanol) to reduce autofluorescence, particularly in tissues like kidney that contain lipofuscin. Blocking should include 5-10% normal serum from the secondary antibody species, plus 1-2% BSA to reduce non-specific binding.

What methodological approaches enable quantitative analysis of CLCNKA expression in disease models?

Quantitative analysis of CLCNKA expression in disease models requires rigorous methodological approaches to ensure reproducibility and accuracy. Western blot quantification remains valuable when performed with appropriate controls - always include housekeeping proteins (β-actin, GAPDH) for normalization and run a dilution series of a reference sample to establish linearity of signal detection . Digital image acquisition and densitometry software (ImageJ, Image Studio) should be used with settings that prevent pixel saturation.

For tissue expression patterns, quantitative immunohistochemistry employing standardized staining protocols and digital image analysis permits spatial assessment of expression changes. Utilize whole slide scanning technologies with automated detection of positive staining intensity and distribution. For pixel-based quantification, define consistent thresholds for positivity and report results as H-scores or percentage of positive area.

RT-qPCR provides complementary data on CLCNKA transcript levels - design primers spanning exon-exon junctions to prevent genomic DNA amplification, and validate primer efficiency using standard curves. Normalize expression to multiple reference genes selected for stability in your specific tissue and disease context.

For the most rigorous quantification, consider mass spectrometry-based proteomics using labeled reference peptides specific to CLCNKA. This approach enables absolute quantification of protein levels independent of antibody affinity variations. Targeted approaches like parallel reaction monitoring (PRM) or selected reaction monitoring (SRM) offer increased sensitivity for low-abundance membrane proteins like CLCNKA.

When comparing disease versus control samples, employ sufficient biological replicates (minimum n=5 per group) and appropriate statistical approaches that account for data distribution characteristics. Report fold changes with confidence intervals rather than just p-values to convey biological significance.

What recent findings have emerged regarding CLCNKA function and its potential role in disease pathogenesis?

Recent research has expanded our understanding of CLCNKA's physiological significance and disease associations. CLCNKA functions as a chloride channel primarily expressed in the kidney's thick ascending limb of Henle's loop and distal convoluted tubule, where it plays a critical role in chloride reabsorption and consequently influences electrolyte homeostasis and blood pressure regulation. The channel typically functions as a heterodimer with Barttin, an essential β-subunit that facilitates proper membrane trafficking and channel function.

Studies using CLCNKA antibodies have identified altered expression patterns in several pathological conditions. In hypertension models, researchers have observed dysregulated CLCNKA expression, suggesting potential involvement in blood pressure control mechanisms. The channel's expression has been documented in multiple human tissues beyond the kidney, including placenta, testis, skin, spleen, lung, and ovary , though its functional significance in these tissues remains under investigation.

Genetic research has identified several CLCNKA mutations associated with Bartter syndrome type III when occurring in conjunction with mutations in the closely related CLCNKB gene. These findings highlight the potential compensatory relationship between these homologous channels. Pharmacological studies have explored compounds that modulate CLCNKA activity, with potential therapeutic applications for conditions involving electrolyte disturbances.

Methodologically, recent advances in cryo-electron microscopy have provided structural insights into the CLC family of proteins, enhancing our understanding of CLCNKA's molecular architecture and gating mechanisms. This structural information provides a foundation for rational drug design targeting this channel in various pathophysiological contexts.