KCNK3 Antibody, HRP conjugated

What is KCNK3 and why is it significant in research?

KCNK3 (also known as TASK-1) is a member of the superfamily of potassium channel proteins containing two pore-forming P domains. It functions as an outwardly rectifying channel sensitive to extracellular pH changes and is inhibited by acidification. As an acid-sensitive potassium channel, KCNK3 is activated by anesthetics including halothane and isoflurane . The significance of KCNK3 in research spans multiple areas, particularly in pulmonary arterial hypertension (PAH) where its expression is notably down-regulated in the right ventricle of PAH patients and in experimental pulmonary hypertension models . This makes KCNK3 a crucial target for understanding ion channel dysfunction in pathological conditions. The channel's role in membrane potential regulation and cellular responses to both physiological and pathological stimuli positions it as an important subject for neuroscience and cardiovascular research .

What applications are recommended for KCNK3 Antibody, HRP conjugated?

The recommended applications for KCNK3 Antibody, HRP conjugated vary by manufacturer and specific antibody formulation. Based on available product information, this antibody is typically suitable for Western Blot analysis at dilutions ranging from 1:100-1000, immunohistochemistry on paraffin-embedded tissues (IHC-P) at dilutions of 1:100-500 , and enzyme-linked immunosorbent assay (ELISA) . The horseradish peroxidase (HRP) conjugation enables direct detection without secondary antibodies, facilitating enhanced chemiluminescence (ECL) detection systems . When designing experiments with this antibody, researchers should conduct preliminary optimization experiments to determine the optimal dilution for their specific tissue or cell type and detection system.

What are the recommended storage and handling conditions for KCNK3 Antibody, HRP conjugated?

To maintain optimal activity of KCNK3 Antibody, HRP conjugated, proper storage and handling are essential. According to product specifications, the antibody should be stored at -20°C or -80°C upon receipt . The formulation typically includes preservatives such as 0.03% Proclin 300 and stabilizers like 50% glycerol in a buffered solution (0.01M PBS, pH 7.4) . Researchers should avoid repeated freeze-thaw cycles as these can degrade the antibody and reduce its effectiveness. Working aliquots should be prepared to minimize freeze-thaw cycles. When handling the antibody, maintain aseptic conditions and use sterile techniques to prevent contamination. The liquid form of the antibody should be maintained at the recommended temperature when not in use, and exposure to light should be minimized, particularly during long-term storage.

How can KCNK3 Antibody, HRP conjugated be used to investigate heteromeric channel associations?

Investigating heteromeric channel associations requires sophisticated biochemical approaches where KCNK3 Antibody, HRP conjugated serves as a valuable tool. For coimmunoprecipitation studies examining KCNK3 (TASK-1) association with other channel subunits like TASK-3, researchers can implement protocols similar to those described in previous studies . In such experiments, cell lysates from systems expressing both channel subunits are immunoprecipitated using anti-KCNK3 antibodies. The precipitated proteins are then separated by SDS-PAGE and immunoblotted with the HRP-conjugated KCNK3 antibody and antibodies against potential interacting partners. The direct HRP conjugation eliminates cross-reactivity issues when detecting rabbit-derived primary antibodies. Control experiments should include preimmune rabbit IgG precipitations and single-transfected cells to confirm specificity. Additionally, membrane-associated proteins like transferrin receptor can serve as negative controls to verify the specificity of the interaction . This approach allows researchers to determine whether KCNK3 forms heteromeric channels with other potassium channel subunits in various experimental systems.

What methodological approaches should be used when studying KCNK3 expression changes in pulmonary arterial hypertension models?

When investigating KCNK3 expression changes in pulmonary arterial hypertension (PAH) models, researchers should implement a comprehensive methodological approach combining molecular, cellular, and physiological techniques. Recent studies have shown that KCNK3 expression is down-regulated in pulmonary arterial smooth muscle cells (PASMCs) from idiopathic PAH patients and in multiple experimental models including monocrotaline-induced (MCT-PH), Sugen 5416/hypoxia-induced, and hypoxia-induced pulmonary hypertension .

A robust experimental design should include:

• Transcript level analysis: Quantitative RT-PCR to measure KCNK3 mRNA levels in lung tissue and isolated PASMCs, with normalization to multiple housekeeping genes.

• Protein expression assessment: Western blotting using KCNK3 Antibody, HRP conjugated with careful optimization of protein extraction protocols to preserve membrane proteins. Immunohistochemistry should be performed to localize expression changes within specific pulmonary vascular structures.

• Functional studies: Electrophysiological techniques to assess KCNK3 channel activity, membrane potential measurements using fluorescent dyes like DiBAC₄(3), and calcium imaging with fura-2 to correlate channel expression with functional consequences .

• Intervention studies: Therapeutic approaches targeting KCNK3 expression, such as miR-138-5p inhibition, which has been shown to restore KCNK3 expression in experimental models .

• Molecular pathway analysis: Investigation of regulatory mechanisms affecting KCNK3 expression, including miRNA regulation and signaling pathways like JNK that may connect KCNK3 dysfunction to vascular remodeling .

Proper controls should include comparison to non-PAH samples and selective channel blockers to confirm specificity of observed effects.

What controls and validation steps are necessary when using KCNK3 Antibody, HRP conjugated in Western blot analysis?

Rigorous validation is essential when using KCNK3 Antibody, HRP conjugated for Western blot analysis. Based on established research practices, the following controls and validation steps should be implemented:

• Positive controls: Include lysates from cells or tissues known to express KCNK3, such as neuronal tissues or transfected cells expressing tagged KCNK3 constructs. This confirms the antibody can detect the target protein under your experimental conditions .

• Negative controls: Analyze samples from KCNK3 knockout models or cells where KCNK3 is not expressed. Alternatively, use siRNA knockdown samples to demonstrate signal reduction corresponding to decreased protein levels.

• Peptide competition assay: Preincubate the antibody with excess KCNK3 antigenic peptide (10× concentration) before application to the blot. Specific immunoreactivity should be blocked by this preabsorption .

• Molecular weight verification: KCNK3 typically appears as multiple bands representing different glycosylation states and/or oligomeric forms. Compare the observed pattern with published literature.

• Loading controls: Include appropriate loading controls such as β-actin, GAPDH, or for membrane proteins, Na⁺/K⁺ ATPase or transferrin receptor .

• Cross-reactivity assessment: Verify the antibody does not detect closely related potassium channels by testing against lysates expressing other K2P family members.

• Comparison with other detection methods: When possible, confirm results using antibodies targeting different epitopes of KCNK3 or using tagged constructs detected with anti-tag antibodies .

These validation steps ensure that observed signals genuinely represent KCNK3 protein and are not artifacts or non-specific interactions.

How can KCNK3 Antibody, HRP conjugated be utilized in miRNA regulation studies of pulmonary hypertension?

KCNK3 Antibody, HRP conjugated provides a valuable tool for investigating miRNA-mediated regulation of KCNK3 in pulmonary hypertension research. Recent studies have identified miR-138-5p as a key regulator of KCNK3 expression in pulmonary arterial hypertension (PAH) . To effectively utilize this antibody in miRNA regulation studies, researchers should implement the following approach:

• Expression correlation analysis: After miRNA modulation (overexpression or inhibition), use KCNK3 Antibody, HRP conjugated in Western blot analyses to quantify protein level changes. This provides direct evidence of post-transcriptional regulation by the miRNA of interest.

• In vivo validation: Following in vivo miRNA modulation (e.g., nebulization-based delivery of anti-miR-138-5p to rats with established MCT-PH), harvest lung tissues for protein extraction and subsequent Western blotting with KCNK3 Antibody, HRP conjugated to assess restoration of KCNK3 expression .

• Cellular localization studies: Perform immunohistochemistry or immunofluorescence on tissue sections using the antibody to determine if miRNA regulation affects KCNK3 expression in specific cell types within the pulmonary vasculature.

• Mechanistic pathway analysis: Combine KCNK3 protein detection with assessment of downstream effectors to establish functional consequences of miRNA-mediated KCNK3 regulation, such as changes in JNK phosphorylation levels .

• Target validation: Use reporter assays with wild-type and mutated KCNK3 3'UTR constructs in conjunction with protein detection to confirm direct targeting by the miRNA of interest.

This integrated approach enables researchers to establish causal relationships between miRNA dysregulation, KCNK3 expression changes, and pathophysiological consequences in pulmonary hypertension models.

What techniques can be used to differentiate between KCNK3 (TASK-1) and other closely related potassium channel proteins?

Differentiating between KCNK3 (TASK-1) and other closely related potassium channel proteins requires a combination of biochemical, molecular, and electrophysiological approaches. When using KCNK3 Antibody, HRP conjugated for this purpose, consider implementing these techniques:

• Epitope mapping and antibody specificity: The KCNK3 Antibody, HRP conjugated should be raised against carefully selected peptide sequences unique to KCNK3. For example, antibodies targeting the C-terminus (residues 278-379) of human KCNK3 provide specificity . Verify the antibody's specificity by testing against lysates containing other K2P family members, particularly TASK-3 (KCNK9) which shares significant homology.

• Western blot pattern analysis: KCNK3 displays characteristic banding patterns that can be distinguished from other K2P channels. Compare the molecular weight and pattern of bands with published data for KCNK3 and other family members.

• Heterologous expression systems: Utilize cells transfected with specific K2P channel members as positive and negative controls. The antibody should specifically detect KCNK3-transfected cells but not cells expressing other channel subunits .

• Immunoprecipitation followed by mass spectrometry: When analyzing complex samples, perform immunoprecipitation with the KCNK3 antibody followed by mass spectrometry identification to confirm the presence of KCNK3-specific peptides and absence of peptides from other K2P channels.

• Pharmacological profiling: Combine immunodetection with functional studies using selective channel modulators (e.g., sensitivity to acidification, halothane, isoflurane) to correlate protein expression with channel-specific functional properties .

• siRNA knockdown validation: Perform siRNA knockdown of KCNK3 and verify that the antibody signal is specifically reduced without affecting signals from other K2P channels.

This multi-faceted approach ensures that the detected signals truly represent KCNK3 rather than related potassium channel proteins, which is crucial for accurate interpretation of experimental results.

What are the optimal dilution ratios for different applications of KCNK3 Antibody, HRP conjugated?

Determining the optimal dilution ratios for KCNK3 Antibody, HRP conjugated is critical for achieving specific signals while minimizing background. Based on product specifications and research protocols, the following application-specific dilution ranges are recommended:

• Western Blot analysis: The recommended dilution range is 1:100-1000 . For initial optimization, test a dilution series (e.g., 1:100, 1:250, 1:500, 1:1000) with positive control samples. Optimal dilution depends on protein expression levels, detection system sensitivity, and blocking conditions. When using enhanced chemiluminescence (ECL), start with more dilute antibody solutions and adjust based on signal intensity.

• Immunohistochemistry on paraffin-embedded tissues (IHC-P): The recommended dilution range is 1:100-500 . Optimization should include antigen retrieval method testing, as membrane proteins like KCNK3 may require specific retrieval protocols. Begin with a middle dilution (1:250) and adjust based on signal-to-noise ratio.

• ELISA: Typically requires more concentrated antibody preparations. Start with 1:100 dilution and optimize based on standard curves with known concentrations of recombinant KCNK3 protein.

• Immunoprecipitation: When using for immunoprecipitation followed by Western blot analysis, start with approximately 2-5 μg of antibody per 500 μg of total protein in lysate .

For all applications, perform parallel negative controls (omitting primary antibody, using non-specific IgG) to assess background levels. Optimization experiments should include varying antibody concentrations, incubation times, and washing conditions to achieve the highest signal-to-noise ratio for your specific experimental system.

How can researchers overcome common challenges when using KCNK3 Antibody, HRP conjugated in complex tissue samples?

Working with KCNK3 Antibody, HRP conjugated in complex tissue samples presents several challenges that can be addressed through methodological refinements:

• High background signal: This common issue can be mitigated by:

  • Using optimized blocking solutions (5% milk or BSA in TBS)

  • Extending blocking time (2-3 hours at room temperature or overnight at 4°C)

  • Increasing washing duration and number of washes

  • Adding 0.1-0.3% Triton X-100 to reduce non-specific membrane interactions

  • Using specialized blocking reagents containing both proteins and detergents

• Low or absent signal: To enhance KCNK3 detection in tissues with low expression:

  • Optimize protein extraction methods specifically for membrane proteins

  • Use specialized membrane protein extraction buffers containing non-ionic detergents

  • Extend primary antibody incubation (overnight at 4°C)

  • Consider signal amplification systems compatible with HRP

  • Avoid reducing agents that may affect HRP activity during sample preparation

• Non-specific bands: To improve specificity:

  • Perform peptide competition assays to identify specific bands

  • Compare band patterns with transfected cells expressing KCNK3

  • Use gradient gels to better resolve proteins of similar molecular weights

  • Adjust transfer conditions to optimize transfer of membrane proteins

• Tissue-specific challenges: For pulmonary tissues specifically:

  • Implement specialized fixation protocols that preserve membrane proteins

  • Use antigen retrieval methods optimized for ion channels

  • Consider using fresh-frozen tissues rather than paraffin-embedded samples for some applications

  • When using lung tissues, account for high endogenous peroxidase activity by including additional blocking steps

• Quantification issues: For accurate quantification:

  • Use internal loading controls appropriate for membrane proteins

  • Implement standard curves with recombinant proteins when possible

  • Use image analysis software with background subtraction capabilities

  • Consider multiple exposure times to ensure linearity of signal

These methodological refinements can significantly improve the reliability and sensitivity of KCNK3 detection in complex tissue samples from disease models such as pulmonary hypertension.

How is KCNK3 Antibody, HRP conjugated being used to investigate the relationship between KCNK3 and developmental disorders?

Recent research has begun to explore the relationship between KCNK3 and developmental disorders, with KCNK3 Antibody, HRP conjugated serving as a key investigative tool. While the search results mention gain-of-function mutations in KCNK3 being associated with a developmental disorder , comprehensive methodological approaches using antibody-based detection can further elucidate this relationship. Researchers investigating this emerging area should consider the following approaches:

• Expression profiling during development: Use KCNK3 Antibody, HRP conjugated in Western blot analysis of tissue samples collected at different developmental stages to establish normal temporal expression patterns. This provides a baseline against which pathological changes can be compared.

• Cellular and subcellular localization studies: Employ immunohistochemistry and immunofluorescence techniques to determine the spatial distribution of KCNK3 in developing tissues, with particular attention to neuronal structures where potassium channels play crucial developmental roles.

• Mutation impact assessment: For identified KCNK3 mutations, express wild-type and mutant constructs in cell models, then use the antibody to compare expression levels, protein stability, and subcellular localization of normal versus mutant channels.

• Interactome analysis: Use KCNK3 Antibody, HRP conjugated in co-immunoprecipitation studies followed by mass spectrometry to identify developmental stage-specific interaction partners that may be disrupted in pathological conditions.

• Comparative studies in model organisms: Apply the antibody in studies of model organisms (where cross-reactivity exists) carrying analogous mutations to those identified in human developmental disorders.

This methodological framework enables researchers to establish mechanistic links between KCNK3 dysfunction and developmental abnormalities, potentially identifying new therapeutic targets for developmental disorders with ion channel etiology.

What are the latest methodological advances in utilizing KCNK3 Antibody, HRP conjugated for therapeutic target validation?

The validation of KCNK3 as a therapeutic target has advanced significantly, with KCNK3 Antibody, HRP conjugated playing a crucial role in these methodologies. Recent research, particularly in pulmonary hypertension models, has demonstrated several innovative approaches:

• miRNA-based therapeutic validation: Studies have employed KCNK3 Antibody, HRP conjugated to demonstrate that in vivo inhibition of miR-138-5p restores KCNK3 expression in monocrotaline-induced pulmonary hypertension models, correlating with improved hemodynamics and reduced vascular remodeling . This methodological approach provides direct evidence linking KCNK3 restoration to therapeutic outcomes.

• Combinatorial target assessment: Advanced methodologies now combine KCNK3 protein detection with simultaneous assessment of multiple pathway components, such as SLC45A3 and JNK phosphorylation status . This systems biology approach provides a more comprehensive understanding of how KCNK3-targeted interventions affect disease-relevant pathways.

• In situ validation in patient-derived cells: Researchers are using KCNK3 Antibody, HRP conjugated to assess channel expression in patient-derived pulmonary arterial smooth muscle cells (PASMCs) before and after therapeutic interventions, providing translational evidence for target engagement .

• Correlation of molecular and functional endpoints: Modern methodologies correlate antibody-based detection of KCNK3 protein levels with functional readouts such as membrane potential measurements, calcium imaging, and proliferation/migration assays . This multi-parameter assessment strengthens the causal relationship between KCNK3 modulation and functional outcomes.

• Targeted delivery system evaluation: KCNK3 Antibody, HRP conjugated is being used to verify target engagement following administration of therapeutics via specialized delivery systems, such as nebulization-based delivery of miRNA inhibitors to the lungs .

These methodological advances provide robust validation frameworks that strengthen the position of KCNK3 as a therapeutic target in conditions characterized by ion channel dysfunction, particularly pulmonary arterial hypertension.