KCNK17 Antibody

What is KCNK17 and why is it important in physiological research?

KCNK17 (also known as TALK-2 or TASK-4) is a member of the potassium channel subfamily K, containing two pore-forming P domains. It functions as an open rectifier that primarily passes outward current under physiological K+ concentrations and is activated at alkaline pH . Physiologically, KCNK17 channels are present in the cardiac conduction system where they regulate action potential duration and beating frequency of cardiac myocytes. The channel conducts voltage-dependent outward rectifying currents through an 'ion flux gating' mechanism where outward ion flow opens the gate . KCNK17's importance in research stems from its involvement in cardiac function and potential role as a diagnostic/prognostic biomarker in conditions such as hepatocellular carcinoma .

What should researchers consider when selecting a KCNK17 antibody for their experiments?

When selecting a KCNK17 antibody, researchers should consider:

• Target epitope location: Antibodies targeting different regions (N-terminal vs. C-terminal) may yield different results. For instance, antibodies targeting the N-terminal region (like ab198043) may be preferable for certain applications .

• Validated applications: Confirm the antibody has been validated for your specific application:

  • Western blot concentrations typically range from 0.04-0.4 μg/ml to 1:500-1:1000 dilutions

  • IHC-P typically requires 1:50-1:200 dilutions

  • For fluorescent applications, consider pre-conjugated antibodies or custom conjugation services

• Species reactivity: Most commercial KCNK17 antibodies react with human samples, with limited cross-reactivity to mouse or rat . Verify your specific research model is covered.

• Clonality: Polyclonal antibodies offer broader epitope recognition but may have higher batch-to-batch variability compared to monoclonals .

• Validation data: Review Western blot and IHC images provided by manufacturers to ensure the antibody detects the appropriate band size (~37 kDa) and expected tissue localization patterns .

What are the optimal protocols for detecting KCNK17 using Western blot analysis?

For optimal Western blot detection of KCNK17:

Sample preparation and electrophoresis:

• Use 8% SDS-PAGE for optimal protein separation

• Load 40-50 μg of total protein per lane for cell lysates

• Include positive controls such as KCNK17 overexpression lysates and negative controls (vector-only transfected cells)

Transfer and detection conditions:

• Recommended antibody concentrations:

  • Primary antibody: 0.04-0.4 μg/ml or 1:300-1:1000 dilution

  • Secondary antibody: Follow manufacturer recommendations for HRP-conjugated antibodies

Expected results:

• KCNK17 appears as a band at approximately 37 kDa

• Exposure time should be optimized (typical starting point: 10 seconds)

Troubleshooting:

• If multiple bands appear, consider:

  • Adding protease inhibitors to prevent degradation

  • Using fresh samples to minimize proteolysis

  • Performing validation using overexpression systems

How should immunohistochemistry protocols be optimized for KCNK17 detection in tissue samples?

For effective KCNK17 immunohistochemistry:

Tissue preparation:

• Formalin fixation and paraffin embedding is suitable for KCNK17 detection

• Optimal section thickness: 4-6 μm

Antigen retrieval:

• Heat-induced epitope retrieval (HIER) at pH 6 is recommended

Antibody dilution and incubation:

• Typical dilution range: 1:50-1:200

• Incubation: Overnight at 4°C for optimal signal-to-noise ratio

Detection systems:

• Both DAB and fluorescent detection systems are suitable

• For fluorescent detection, consider using pre-conjugated antibodies (Alexa Fluor 488, etc.)

Expected staining patterns:

• Nuclear positivity has been observed in testicular seminiferus ducts

• Cytoplasmic/membrane staining in other tissues

Controls:

• Include tissues with known KCNK17 expression (cardiac tissue, testis)

• Use negative controls (primary antibody omission or non-immune IgG)

What approaches can be used to verify the specificity of KCNK17 antibodies?

To verify KCNK17 antibody specificity:

Overexpression systems:

• Compare control (vector-only transfected) versus KCNK17-overexpressing cells (e.g., HEK293T cells)

• Use recombinant KCNK17 protein as a positive control

Knockdown/knockout validation:

• Use siRNA/shRNA against KCNK17 to reduce expression

• CRISPR/Cas9-mediated knockout as gold standard for specificity verification

Peptide competition assays:

• Pre-incubate antibody with immunizing peptide to block specific binding

• Use peptides corresponding to the immunogen sequence (e.g., amino acids 271-320 for some antibodies)

Multiple antibody validation:

• Compare staining patterns using antibodies targeting different epitopes

• Consistent results across multiple antibodies suggest higher specificity

Enhanced validation approaches:

• Recombinant expression validation as indicated for some commercial antibodies

• Protein array testing (some antibodies are tested against arrays of 364 human recombinant protein fragments)

What are the considerations for using fluorescently conjugated KCNK17 antibodies?

When using fluorescently conjugated KCNK17 antibodies:

Available conjugates:

• Multiple fluorophore options exist, including:

  • Alexa Fluor series (350, 488, 555, 594, 647, 680, 700, 750)

  • Traditional dyes (FITC, TRITC, Cy3, Cy5)

  • iFluor series (multiple wavelengths available)

Dilution requirements:

• For Alexa Fluor 488 conjugated antibodies: typical dilutions range from 1:50-1:250

Application optimization:

• Direct conjugation eliminates secondary antibody cross-reactivity issues

• Useful for multi-color immunofluorescence when antibodies from the same host species are needed

Custom conjugation options:

• Multiple services offer custom conjugation of unconjugated KCNK17 antibodies

• Protein conjugates (HRP, alkaline phosphatase, streptavidin)

• Small molecule conjugates (biotin)

Storage considerations:

• Protect from light to prevent photobleaching

• Store according to manufacturer recommendations (typically -20°C)

• Avoid repeated freeze-thaw cycles

How has KCNK17 been implicated in cardiac pathophysiology?

KCNK17 plays significant roles in cardiac function with important disease implications:

Gain-of-function mutation:

• A heterozygous mutation in KCNK17 (c.262G>A resulting in p.Gly88Arg) has been identified in patients with severe cardiac conduction disorders

• This mutation occurs in the first extracellular pore loop of the channel and results in:

  • Threefold increased currents through mutant channels

  • Unchanged surface expression, indicating enhanced conductivity

  • Dominant-active effect when co-expressed with wild-type channels

Cardiac conduction system:

• KCNK17 expression in the cardiac conduction system contributes to:

  • Regulation of action potential duration

  • Modulation of cardiac myocyte beating frequency

Clinical significance:

• The gain-of-function mutation was found alongside an SCN5A mutation (c.3963+1G>A) in a patient with severe cardiac phenotype

• Multiple pathogenicity prediction tools rated the p.Gly88Arg mutation as "damaging"

• The glycine residue at position 88 is highly conserved across orthologous channels

This research suggests KCNK17 antibodies are valuable tools for investigating cardiac channelopathies and potential therapeutic targets in cardiac conduction disorders.

What is KCNK17's role in hepatocellular carcinoma and how can antibodies help study this relationship?

KCNK17 has emerging significance in hepatocellular carcinoma (HCC) research:

Expression pattern in HCC:

• KCNK17 shows reduced expression in liver cancer tissues compared to healthy liver

• This altered expression correlates with patient prognosis

Diagnostic and prognostic potential:

• KCNK17, along with other KCNK family members (KCNK2, KCNK9, KCNK15), serves as potential diagnostic and prognostic biomarkers for HCC

• Expression levels can be used to stratify patient risk and predict outcomes

Research applications of KCNK17 antibodies:

• Immunohistochemistry to evaluate protein expression in tumor vs. adjacent normal tissues

• Western blot to quantify expression levels

• Tissue microarray analysis for high-throughput screening

Mechanistic investigations:

• Antibodies enable studies of KCNK17's role in tumor suppression mechanisms

• Analysis of downstream signaling pathways affected by KCNK17 downregulation

These findings suggest KCNK17 antibodies are valuable tools for cancer researchers studying ion channel dysregulation in HCC pathogenesis and for developing potential diagnostic markers.

What methodological approaches can be used to study KCNK17 function in complex disease models?

To investigate KCNK17 function in disease models, researchers can employ:

Expression analysis techniques:

• Quantitative immunohistochemistry using validated antibodies at 1:50-1:200 dilutions

• Western blot analysis of tissue or cell lysates (0.04-0.4 μg/ml antibody concentration)

• RT-qPCR to correlate protein expression with mRNA levels

Functional studies:

• Electrophysiology (patch-clamp) to assess channel conductance in native and mutant forms

• Co-immunoprecipitation using KCNK17 antibodies to identify interaction partners

• Proximity ligation assays to detect protein-protein interactions in situ

Genetic manipulation approaches:

• CRISPR/Cas9 gene editing to create disease-relevant mutations (e.g., p.Gly88Arg)

• siRNA/shRNA knockdown to assess loss-of-function effects

• Overexpression of wild-type or mutant KCNK17 using recombinant proteins

Translational research methods:

• Patient sample analysis using tissue microarrays and KCNK17 antibodies

• Correlation of expression with clinical parameters and outcomes

• Drug screening to identify compounds that modulate KCNK17 activity

Imaging approaches:

• Immunofluorescence microscopy using fluorophore-conjugated antibodies

• Live-cell imaging to track channel trafficking and localization

• Super-resolution microscopy for detailed subcellular localization studies

These methodological approaches, centered around validated KCNK17 antibodies, provide researchers with a comprehensive toolkit to investigate this potassium channel's role in complex disease states.

How can researchers address challenges in detecting post-translational modifications of KCNK17?

Detection of KCNK17 post-translational modifications requires specialized approaches:

Antibody selection:

• Most commercial antibodies target unmodified KCNK17

• Consider using modification-specific antibodies if available or developing custom antibodies against known modification sites

Enrichment strategies:

• Immunoprecipitation with KCNK17 antibodies followed by modification-specific detection

• Phospho-enrichment techniques for detecting phosphorylated forms

• Ubiquitin pulldown assays for ubiquitinated KCNK17

Analytical techniques:

• Mass spectrometry following immunoprecipitation to identify unknown modifications

• 2D gel electrophoresis to separate modified forms prior to Western blotting

• Phos-tag gels for improved separation of phosphorylated species

Validation approaches:

• Treatment with modification-removing enzymes (phosphatases, deubiquitinases)

• Site-directed mutagenesis of putative modification sites

• In vitro modification assays to generate positive controls

Emerging tools:

• Proximity labeling approaches to identify contextual modifications

• Single-molecule imaging techniques to study modification dynamics

• Nanobody-based approaches for detection of modifications in living cells

Understanding KCNK17 post-translational modifications may provide insights into channel regulation and identify novel therapeutic targets in associated diseases.

What emerging technologies are enhancing the utility of KCNK17 antibodies in research?

Several technological advances are improving KCNK17 antibody applications:

Advanced conjugation chemistry:

• Expanded spectrum of fluorophore options including:

  • Alexa Fluor series (350-750 nm)

  • iFluor series with superior brightness and photostability

  • mFluor series optimized for specific excitation sources

Multiplex detection systems:

• Tandem conjugates enabling multi-parameter flow cytometry

• Spectral imaging platforms for simultaneous detection of multiple targets

• Sequential immunostaining protocols for co-localization studies

Enhanced validation approaches:

• Recombinant expression validation

• Protein array testing against hundreds of human proteins

• CRISPR/Cas9 knockout validation systems

Single-cell analysis:

• Mass cytometry (CyTOF) compatible antibody conjugates

• Single-cell Western blot technologies

• Imaging mass cytometry for tissue section analysis

In vivo applications:

• Near-infrared (NIR) conjugates for in vivo imaging

• Targeted therapeutics using antibody-drug conjugates

• Intrabody approaches for tracking KCNK17 in living cells