KCNJ13 Antibody

What is KCNJ13 and what are its alternative names in scientific literature?

KCNJ13 encodes the potassium voltage-gated channel subfamily J member 13 protein in humans, an inward-rectifying potassium ion channel with a molecular weight of approximately 40.5 kilodaltons. In scientific literature, KCNJ13 is also known by several alternative names:

• Kir1.4

• KIR7.1

• SVD

• LCA16

• Inward rectifier potassium channel 13

• Inward rectifier K(+) channel Kir7.1

The gene encodes a protein that plays critical roles in maintaining ion homeostasis in various tissues, particularly in tracheal smooth muscle cells and retinal pigment epithelium .

What criteria should be considered when selecting a KCNJ13 antibody for research applications?

When selecting a KCNJ13 antibody, researchers should carefully evaluate multiple parameters to ensure experimental success:

For specialized applications like studying KCNJ13 in living cells, antibodies targeting extracellular epitopes (such as AA 80-94) offer distinct advantages as they can detect the protein without cell permeabilization .

What is the tissue-specific expression pattern of KCNJ13 during development?

KCNJ13 exhibits distinct spatiotemporal expression patterns during development:

• Embryonic stage E13.5-E14.5: KCNJ13 is primarily detected in tracheal smooth muscle, but not in SOX9+ mesenchymal cells

• After E15.5: Expression extends to a subset of tracheal epithelial cells

• E16.5 and later: KCNJ13 mRNA is detected in lung epithelial cells

• Mature tissues: Expression localizes to epithelial cells of bronchioles and alveolar type II cells

This dynamic expression pattern suggests stage-specific roles during organ development, particularly in the respiratory system .

What controls are essential when validating KCNJ13 antibodies for Western blot experiments?

A robust validation strategy for KCNJ13 antibodies should include multiple controls:

• Positive tissue controls: Samples from tissues known to express KCNJ13 (tracheal smooth muscle, retinal pigment epithelium)

• Cell line panels: Various cell lines with different KCNJ13 expression levels (recommended dilution 1:500-1:2000)

• Knockout/knockdown controls: CRISPR/Cas9-generated KCNJ13 knockout samples or siRNA knockdowns

• Peptide competition: Pre-incubation of antibody with immunizing peptide should abolish specific signal

• Loading controls: Housekeeping proteins (β-actin, GAPDH) to normalize protein loading

• Multiple antibodies comparison: Using antibodies targeting different epitopes of KCNJ13

The optimal antibody concentration should be determined empirically, with recommended starting ranges of 1:500-1:2000 for Western blot applications .

How can CRISPR/Cas9 technology be implemented to generate KCNJ13 knockout cell models?

CRISPR/Cas9 system can be effectively used to generate KCNJ13 knockout models by following these methodological steps:

• gRNA design: Design guide RNAs targeting specific sites in the KCNJ13 gene. For complete knockout, target sequences near the start codon and 3'UTR to delete most of the gene

• Specificity screening: Ensure target sequences show perfect matches with intended sites by checking databases. Select sequences with minimal off-target effects

• gRNA construction: Anneal crRNA (complementary to target sequences) with tracrRNA according to manufacturer protocols

• Delivery method: Transfect cells (e.g., hiPSCs) with Cas9 protein and gRNA complexes

• Clone isolation: Select and expand single cell-derived clones

• Validation: Confirm knockout through genomic PCR, sequencing, and protein expression analysis (Western blot, immunostaining)

• Functional testing: Assess phenotypic changes in knockout cells compared to wildtype

For studying KCNJ13 in retinal contexts, human-induced pluripotent stem cells can be differentiated into retinal pigment epithelial cells after CRISPR/Cas9 modification .

What immunohistochemistry protocols are most effective for detecting KCNJ13 in tissue sections?

For optimal detection of KCNJ13 in tissue sections, consider this methodological approach:

• Tissue preparation:

  • Fix tissues in 4% paraformaldehyde (PFA) for 24 hours

  • Process and embed in paraffin or OCT compound for frozen sections

  • Cut sections at 5-8 μm thickness

• Antigen retrieval:

  • Heat-induced epitope retrieval in citrate buffer (pH 6.0) for 20 minutes

  • Allow to cool to room temperature

• Blocking and permeabilization:

  • Block with 5-10% normal serum (match to secondary antibody host) with 0.1-0.3% Triton X-100

  • Incubate for 1 hour at room temperature

• Primary antibody incubation:

  • Use anti-KCNJ13 antibody at dilutions ranging from 1:100-1:500

  • Incubate overnight at 4°C in a humidified chamber

• Detection system:

  • Fluorescent: Use appropriate secondary antibodies conjugated to fluorophores

  • Chromogenic: Use HRP/AP-conjugated secondary antibodies with suitable substrates

• Co-staining markers:

  • For developmental studies: Include stage-specific markers

  • For cell-type identification: Include markers for smooth muscle (SMA) or epithelial cells (E-cadherin)

• Controls:

  • Primary antibody omission

  • Isotype control antibodies

  • Known positive tissues (trachea, lung epithelium, retina)

How can KCNJ13 antibodies be used to investigate ion channel physiology in living cells?

KCNJ13 antibodies targeting extracellular epitopes provide valuable tools for studying ion channel physiology in living cells through these advanced applications:

• Real-time surface expression monitoring:

  • Use fluorescently-labeled antibodies targeting the extracellular loop (AA 80-94)

  • Perform time-lapse imaging to track channel movement and clustering

• Electrophysiological studies:

  • Combine patch-clamp recordings with antibody application

  • Monitor changes in channel conductance following antibody binding

• Channel blockade studies:

  • Use antibodies as functional blockers to assess physiological consequences

  • Compare with pharmacological inhibitors like VU590 (50 μM)

• Membrane potential assessment:

  • Apply voltage-sensitive fluorescent dyes like DiBAC4(3)

  • Compare membrane potential in KCNJ13-expressing vs. antibody-blocked cells

• Correlation with cytoskeletal dynamics:

  • Combine with F-actin visualization using phalloidin staining

  • Assess actin organization changes following antibody-mediated channel blockade

This approach is particularly valuable when studying the role of KCNJ13 in smooth muscle cell alignment and polarity, as membrane potential changes impact cytoskeletal organization .

What methodologies can unveil the relationship between KCNJ13 function and downstream signaling pathways?

To investigate KCNJ13's influence on downstream signaling pathways, implement these methodological approaches:

• AKT signaling analysis:

  • Western blot analysis of phosphorylated AKT (p-AKT) levels in wild-type vs. KCNJ13-mutant cells

  • Use AKT inhibitors (e.g., MK-2206) and activators to modulate signaling

  • Assess rescue of phenotypes through pharmacological AKT activation

• Calcium imaging:

  • Employ calcium-sensitive dyes or genetically encoded calcium indicators

  • Monitor calcium flux in response to KCNJ13 modulation

  • Correlate with cytoskeletal reorganization

• Phosphoproteomic analysis:

  • Compare phosphorylation profiles between control and KCNJ13-deficient samples

  • Identify differentially regulated pathways through mass spectrometry

  • Validate key phosphorylation events using phospho-specific antibodies

• Pharmacological manipulation:

  • Apply potassium ionophores (valinomycin, 2 μM) to deplete intracellular potassium

  • Use KCNJ13 inhibitors (VU590, 50 μM) to block channel function

  • Assess effects on actin organization and cell morphology

• Genetic interaction studies:

  • Perform combined knockdown/knockout of KCNJ13 and interacting partners

  • Analyze synergistic or epistatic effects on phenotypes

This multi-faceted approach has revealed that KCNJ13-regulated ion homeostasis influences AKT phosphorylation, which in turn affects actin organization crucial for cellular processes like tracheal tubulogenesis .

How can researchers effectively investigate KCNJ13's role in retinal diseases like Leber congenital amaurosis type 16 (LCA16)?

To study KCNJ13's involvement in LCA16, implement this comprehensive research strategy:

• Disease model generation:

  • Create patient-specific induced pluripotent stem cells (iPSCs) from LCA16 patients

  • Generate CRISPR/Cas9 knockouts or specific disease mutations in control iPSCs

  • Differentiate into retinal pigment epithelium (RPE) cells

• Functional assays:

  • Phagocytosis assessment: Measure uptake of fluorescently labeled photoreceptor outer segments

  • Cell alignment analysis: Quantify morphological changes in RPE monolayers

  • Transepithelial resistance: Evaluate barrier function of RPE

• Molecular characterization:

  • Transcriptomic profiling: Compare gene expression between mutant and control RPE

  • Proteomic analysis: Identify changes in protein expression and modification

  • Interactome mapping: Define KCNJ13 interaction partners in RPE

• Therapeutic screening:

  • Gene therapy approaches: Test AAV-mediated KCNJ13 delivery

  • Small molecule screening: Identify compounds that restore ion homeostasis

  • Pharmacological modulation: Target downstream effectors (e.g., AKT pathway)

• In vivo validation:

  • Develop KCNJ13 mutant mouse models

  • Perform electroretinography to assess retinal function

  • Analyze retinal morphology and RPE integrity

This integrated approach will advance understanding of how KCNJ13 mutations lead to retinal dysfunction and identify potential therapeutic strategies for LCA16 .

What strategies can resolve inconsistent KCNJ13 antibody performance across different experimental platforms?

When facing inconsistent KCNJ13 antibody performance, implement this systematic troubleshooting approach:

For particularly challenging applications, consider using genetic tagging approaches (e.g., CRISPR knock-in of epitope tags) as an alternative to antibody-based detection .

How should researchers interpret unexpected molecular weight bands when detecting KCNJ13 by Western blot?

When encountering unexpected bands in Western blot for KCNJ13, apply this analytical framework:

• Expected KCNJ13 band size: The canonical form should appear at approximately 40.5 kDa

• Higher molecular weight bands (60-100 kDa):

  • Potential post-translational modifications (glycosylation, SUMOylation)

  • Incomplete denaturation leading to dimeric/multimeric forms

  • Verification strategy: Treat samples with deglycosylation enzymes or stronger reducing agents

• Lower molecular weight bands (15-35 kDa):

  • Proteolytic degradation products

  • Alternative splice variants

  • Verification strategy: Add additional protease inhibitors; compare with mRNA expression data

• Confirmation approaches:

  • Peptide competition: Pre-incubate antibody with immunizing peptide

  • siRNA knockdown: Compare band intensity in control vs. KCNJ13 knockdown samples

  • Tissue panel: Compare expression across multiple tissues with known KCNJ13 expression

  • Multiple antibodies: Test antibodies targeting different epitopes

• Alternative detection methods:

  • Immunoprecipitation followed by mass spectrometry

  • RT-PCR to identify potential splice variants

Careful interpretation of these patterns can provide valuable insights into tissue-specific processing or modifications of KCNJ13 .

What specialized protocols can enhance detection of low-abundance KCNJ13 in challenging samples?

For detecting low-abundance KCNJ13 in challenging samples, implement these specialized protocols:

• Enhanced protein extraction:

  • Use specialized membrane protein extraction buffers containing:

    • 1% Triton X-100 or NP-40

    • 0.5% sodium deoxycholate

    • Complete protease inhibitor cocktail

  • Sonicate briefly to enhance membrane protein solubilization

  • Concentrate proteins using TCA precipitation or commercial concentrators

• Signal amplification techniques:

  • Tyramide Signal Amplification (TSA): Enhances immunohistochemistry signal by 10-50 fold

  • Polymer-based detection systems: Provides higher sensitivity than standard ABC methods

  • Quantum dots: Offer superior photostability and signal intensity for fluorescence detection

• Proximity Ligation Assay (PLA):

  • Enables detection of protein-protein interactions with single-molecule sensitivity

  • Can detect endogenous KCNJ13 interacting with known binding partners

• Sample enrichment approaches:

  • Perform subcellular fractionation to isolate membrane-enriched fractions

  • Use lectin-based enrichment to capture glycosylated KCNJ13

  • Consider immunoprecipitation prior to Western blotting

• Specialized imaging:

  • For tissue sections, use confocal microscopy with spectral unmixing

  • Implement deconvolution algorithms to enhance signal-to-noise ratio

  • Consider super-resolution microscopy for detailed localization studies

These approaches can significantly enhance detection sensitivity for challenging samples with low KCNJ13 expression .

How can KCNJ13 antibodies contribute to understanding the role of ion channels in cytoskeletal organization?

KCNJ13 antibodies offer powerful tools for investigating the emerging field of ion channel-cytoskeleton interactions:

• Membrane potential-cytoskeleton coupling:

  • Use KCNJ13 antibodies to manipulate channel function in living cells

  • Simultaneously monitor membrane potential (DiBAC4(3)) and F-actin organization (phalloidin)

  • Quantify changes in cell shape, polarity, and alignment following antibody application

• Super-resolution co-localization studies:

  • Apply STORM or PALM microscopy to visualize nanoscale associations between KCNJ13 and cytoskeletal elements

  • Use dual-color labeling with KCNJ13 antibodies and cytoskeletal markers

• Live-cell imaging approaches:

  • Combine extracellular epitope-targeting antibodies with cytoskeletal reporters

  • Track dynamic changes in real-time during cellular processes like migration or division

• Mechanistic investigation:

  • Study AKT phosphorylation levels as a molecular link between KCNJ13 function and actin organization

  • Apply pharmacological modulators (VU590, valinomycin) in conjunction with antibody labeling

This research direction has significant implications for understanding developmental processes like tracheal tubulogenesis and may reveal fundamental principles of ion homeostasis in cellular morphogenesis .

What methodological approaches can investigate potential KCNJ13 involvement in cell types beyond current knowledge?

To explore KCNJ13's uncharacterized roles in additional cell types, implement these methodological approaches:

• Comprehensive expression profiling:

  • Perform single-cell RNA sequencing across multiple tissues and developmental stages

  • Validate expression in identified cell populations using KCNJ13 antibodies

  • Create tissue-specific expression atlases through systematic immunohistochemistry

• Conditional knockout models:

  • Generate floxed KCNJ13 alleles for Cre-mediated cell-type specific deletion

  • Analyze phenotypic consequences in previously unexplored cell populations

  • Compare with global knockout phenotypes to identify tissue-specific functions

• Ex vivo functional assays:

  • Develop organ culture systems (similar to tracheal-lung organ cultures)

  • Apply antibodies or inhibitors to assess KCNJ13 function in development

  • Quantify morphological and functional outcomes in diverse tissues

• Human disease association studies:

  • Analyze KCNJ13 variants in patient cohorts with unexplained channelopathies

  • Screen for KCNJ13 mutations in conditions beyond LCA16

  • Develop cellular models to test variant pathogenicity

These approaches may reveal unexpected roles for KCNJ13 in tissues beyond the currently established tracheal smooth muscle, epithelial, and retinal contexts .