KCNS2 Antibody

What is KCNS2 and why is it important in research?

KCNS2, also known as KV9.2, is a potassium voltage-gated channel belonging to the delayed-rectifier subfamily S. This protein plays a crucial role in regulating neuronal excitability and action potential repolarization in various tissues. Research on KCNS2 is particularly important for understanding channel modulation mechanisms in neurological disorders and cardiovascular function. The protein consists of 477 amino acids with a calculated molecular weight of 54 kDa, though it is typically observed at 65-70 kDa in experimental conditions, suggesting post-translational modifications . As a regulatory subunit, KCNS2 does not form functional homomeric channels but modulates the properties of other Kv channels, making it an important research target in electrophysiology.

What are the key differences between various types of KCNS2 antibodies?

KCNS2 antibodies differ primarily in their epitope targeting, host species, clonality, and validated applications:

The choice between these antibodies depends on the experimental design and the specific region of KCNS2 under investigation . Researchers should select antibodies targeting regions not affected by potential splice variants or post-translational modifications relevant to their study.

How does the observed molecular weight of KCNS2 differ from the calculated weight, and why?

• Post-translational modifications, particularly glycosylation of the extracellular domains

• Phosphorylation states that affect protein mobility in SDS-PAGE

• The highly hydrophobic nature of membrane channel proteins, which can bind more SDS and show aberrant migration

• Potential protein-protein interactions that may resist complete denaturation

Researchers should be aware of this discrepancy when interpreting Western blot results and consider using positive controls with known molecular weight patterns to confirm antibody specificity .

What are the optimal protocols for using KCNS2 antibodies in Western blotting?

For optimal Western blotting with KCNS2 antibodies, the following methodology is recommended:

• Sample preparation:

  • Use fresh tissue or cells where KCNS2 is expressed (brain tissue, Y79 cells, or HepG2 cells)

  • Lyse samples in RIPA buffer containing protease inhibitors

  • Include phosphatase inhibitors if phosphorylation states are relevant

• Gel electrophoresis and transfer:

  • Load 20-50 μg of protein per lane

  • Use 8-10% polyacrylamide gels for better resolution of the 65-70 kDa band

  • Transfer to PVDF membranes (preferred over nitrocellulose for hydrophobic proteins)

• Antibody incubation:

  • Block membranes with 5% non-fat milk or BSA in TBST

  • Dilute KCNS2 antibody 1:500-1:2000 in blocking buffer

  • Incubate overnight at 4°C or for 1.5 hours at room temperature

  • Use HRP-conjugated secondary antibodies at 1:3000-1:5000 dilution

• Detection:

  • Enhanced chemiluminescence (ECL) detection is suitable

  • Expect bands at 65-70 kDa for KCNS2

This protocol has been validated with multiple KCNS2 antibodies and consistently produces specific bands in tissue samples known to express the protein.

How can KCNS2 antibodies be effectively used in immunohistochemistry?

For successful immunohistochemistry (IHC) with KCNS2 antibodies, follow these specialized steps:

• Tissue preparation:

  • Fix tissues in 4% paraformaldehyde or 10% neutral buffered formalin

  • Paraffin-embed or prepare frozen sections (10-20 μm thickness)

• Antigen retrieval (critical step):

  • Use TE buffer at pH 9.0 (preferred method)

  • Alternative: citrate buffer at pH 6.0 if TE buffer yields high background

  • Heat-induced epitope retrieval at 95-100°C for 15-20 minutes

• Immunostaining:

  • Block endogenous peroxidases with 3% H₂O₂

  • Block non-specific binding with 5-10% normal serum from secondary antibody host

  • Dilute KCNS2 antibody 1:20-1:200 (optimize for each tissue type)

  • Incubate overnight at 4°C or 1-2 hours at room temperature

  • Use appropriate detection system (HRP-polymer recommended)

  • Develop with DAB and counterstain with hematoxylin

• Controls:

  • Include negative controls (omitting primary antibody)

  • Use tissues with known KCNS2 expression (e.g., human eye tissue) as positive controls

Special consideration: KCNS2 antibodies have been particularly successful in detecting the protein in human eye tissue and brain sections, where the channel plays important physiological roles.

What are the validated methods for using KCNS2 antibodies in immunoprecipitation experiments?

For immunoprecipitation (IP) of KCNS2, the following protocol is recommended based on validated research applications:

• Cell/tissue lysate preparation:

  • Use non-denaturing lysis buffer (e.g., 1% NP-40, 150 mM NaCl, 50 mM Tris-HCl pH 7.5)

  • Include protease and phosphatase inhibitor cocktails

  • Clear lysates by centrifugation (15,000g for 15 minutes at 4°C)

• Pre-clearing step:

  • Incubate lysate with protein A/G beads for 1 hour at 4°C

  • Remove beads by centrifugation to reduce non-specific binding

• Immunoprecipitation:

  • Use 0.5-4.0 μg of KCNS2 antibody per 1.0-3.0 mg of total protein lysate

  • Incubate antibody with lysate overnight at 4°C with gentle rotation

  • Add pre-washed protein A/G beads and incubate for 2-4 hours at 4°C

  • Wash beads 3-5 times with lysis buffer

• Analysis:

  • Elute proteins with SDS sample buffer

  • Analyze by Western blot using another KCNS2 antibody recognizing a different epitope

This method has been successfully used with HepG2 cells expressing endogenous KCNS2 . The IP protocol allows for investigation of protein-protein interactions involving KCNS2, which is particularly relevant for understanding how this regulatory subunit modulates other Kv channels.

What are common issues when detecting KCNS2 and how can they be resolved?

Several technical challenges can arise when working with KCNS2 antibodies, and these can be addressed through specific optimizations:

For Western blot applications specifically, KCNS2 detection has been optimized in Y79 cells using a 1:300 dilution of antibody with room temperature incubation for 1.5 hours . For challenging samples, longer exposure times during chemiluminescent detection may be necessary due to potential low expression levels in some tissues.

How do post-translational modifications affect KCNS2 antibody binding and detection?

Post-translational modifications (PTMs) of KCNS2 can significantly impact antibody recognition and experimental outcomes:

• Phosphorylation:

  • KCNS2 contains multiple serine/threonine phosphorylation sites

  • Phosphorylation can change antibody accessibility to epitopes

  • Solution: When investigating phosphorylation-dependent function, use phosphatase inhibitors in lysates and consider dephosphorylation controls

• Glycosylation:

  • N-linked glycosylation contributes to the higher observed molecular weight

  • Deglycosylation treatments (PNGase F) may alter antibody binding

  • Solution: For studies focusing on core protein, consider enzymatic deglycosylation before detection

• Ubiquitination/SUMOylation:

  • These modifications regulate channel turnover and may appear as higher molecular weight bands

  • Solution: Use deubiquitinating enzyme inhibitors when studying protein degradation pathways

For experiments specifically investigating PTMs, researchers should select KCNS2 antibodies targeting regions distant from known modification sites to ensure detection regardless of modification state . This is particularly important for functional studies correlating channel activity with protein expression levels.

How can researchers validate KCNS2 antibody specificity for their particular experiments?

Validating antibody specificity is critical for reliable KCNS2 research. A comprehensive validation approach includes:

• Positive and negative tissue controls:

  • Positive: Use tissues with known KCNS2 expression (brain, eye, HepG2 cells)

  • Negative: Use tissues with minimal KCNS2 expression or knockout models if available

• Peptide competition assays:

  • Pre-incubate the antibody with excess immunizing peptide

  • Compare detection with and without peptide competition

  • Specific signals should be blocked by the peptide

• Orthogonal detection methods:

  • Correlate protein detection with mRNA expression using qPCR

  • Use multiple antibodies targeting different epitopes of KCNS2

  • Compare results across different detection techniques (WB, IHC, IP)

• siRNA knockdown:

  • Transfect cells with KCNS2-specific siRNA

  • Confirm reduction in signal intensity correlates with knockdown efficiency

For advanced validation, mass spectrometry analysis of immunoprecipitated proteins can provide definitive confirmation of antibody specificity by identifying KCNS2 peptides in the isolated sample . This multi-faceted approach ensures that experimental findings accurately reflect KCNS2 biology rather than artifacts of non-specific antibody interactions.

How can KCNS2 antibodies be used to study protein-protein interactions with other Kv channel subunits?

KCNS2 (Kv9.2) primarily functions as a modulatory subunit that interacts with Kv2 family members. To study these interactions:

• Co-immunoprecipitation strategy:

  • Immunoprecipitate with anti-KCNS2 antibody

  • Probe Western blots with antibodies against Kv2.1 or Kv2.2

  • Alternatively, perform reverse co-IP with Kv2 antibodies

  • Use gentle lysis conditions (1% digitonin or 0.5% NP-40) to preserve interactions

• Proximity ligation assay (PLA):

  • Use KCNS2 antibody paired with Kv2.x antibodies from different host species

  • PLA signal indicates proteins are within 40 nm of each other

  • This technique reveals native interactions in intact cells/tissues

• FRET/BRET analysis with tagged constructs:

  • Verify interactions observed with antibodies using fluorescent protein fusions

  • Correlate antibody-based findings with functional energy transfer data

Research using these approaches has demonstrated that KCNS2 forms heteromeric channels with Kv2.1/Kv2.2, modifying their kinetics and voltage dependence . When designing these experiments, researchers should select antibodies targeting regions not involved in subunit interactions to avoid interfering with the native complex formation.

What are the considerations for using KCNS2 antibodies in studies of channelopathies and neurological disorders?

When investigating KCNS2 in disease contexts, researchers should consider:

• Epitope accessibility in pathological states:

  • Protein conformational changes in disease may affect antibody binding

  • Use multiple antibodies targeting different regions to ensure detection

  • Compare results in control versus disease tissues to assess potential epitope masking

• Expression pattern analysis:

  • Changes in subcellular localization may indicate dysfunction

  • Use confocal microscopy with co-localization markers for detailed analysis

  • Compare membrane versus cytoplasmic fractions in biochemical assays

• Post-translational modification changes:

  • Disease states often alter phosphorylation or glycosylation patterns

  • Consider using phospho-specific antibodies when available

  • Perform 2D gel electrophoresis to separate differently modified forms

• Model-specific validations:

  • Validate antibody performance in each disease model or human sample type

  • Adjust protocols for potentially altered tissue properties (e.g., increased lipid content)

Several studies have implemented these approaches to investigate KCNS2 alterations in epilepsy models and neurodegenerative conditions, where channel dysfunction contributes to altered neuronal excitability . When reporting findings, researchers should clearly document the specific antibody used and validation performed in the disease context to ensure reproducibility.

How can researchers optimize KCNS2 antibody-based techniques for low-expression tissues?

KCNS2 can be challenging to detect in tissues with low expression levels. Advanced techniques to enhance detection include:

• Signal amplification methods:

  • Tyramide signal amplification (TSA) for IHC/IF can increase sensitivity 10-100 fold

  • Use biotin-streptavidin systems for Western blot detection

  • Consider chemiluminescent substrates with extended light emission

• Sample enrichment strategies:

  • Perform subcellular fractionation to concentrate membrane proteins

  • Use immunoprecipitation before Western blotting for detection

  • Employ laser capture microdissection to isolate specific cell populations

• Optimized fixation protocols for IHC:

  • Test different fixatives (paraformaldehyde, Bouin's, zinc-based fixatives)

  • Adjust fixation times to prevent epitope masking

  • Employ multiple antigen retrieval methods (heat, enzymatic, pH variants)

• Detection system enhancements:

  • Use high-sensitivity digital imaging systems

  • Employ computational image analysis for signal quantification

  • Consider newer generation HRP substrates with femtogram detection limits

Researchers successfully detecting KCNS2 in challenging samples have reported that optimizing antibody concentration (1:300 dilution), extending primary antibody incubation time (overnight at 4°C), and employing signal amplification systems significantly improved detection in tissues with naturally low expression levels .

What considerations should be made when using KCNS2 antibodies across different species?

KCNS2 shows considerable sequence conservation across mammals, but species-specific considerations include:

• Epitope conservation analysis:

  • Perform sequence alignment of the antibody epitope region across target species

  • Higher homology in the epitope region predicts better cross-reactivity

  • Test antibodies in samples from each species before main experiments

• Species-specific optimization:

  • Antibody dilutions may need adjustment for different species (typically 1:300-1:2000)

  • Antigen retrieval conditions often require species-specific optimization

  • Secondary antibody selection should account for potential cross-reactivity

• Control selection:

  • Include species-specific positive controls (e.g., brain tissue)

  • Consider recombinant protein controls if native samples are unavailable

  • Validate knockdown/knockout models in the specific species being studied

Available KCNS2 antibodies have demonstrated reactivity with human, mouse, and rat tissues, but sensitivity may vary . For example, antibodies generated against the internal region generally show broader cross-species reactivity than those targeting terminal regions, which may have greater sequence divergence. When working with non-validated species, preliminary testing with multiple antibody dilutions and appropriate controls is essential.

How can KCNS2 antibodies be applied in high-throughput screening methods?

KCNS2 antibodies can be adapted for high-throughput applications through several approaches:

• Automated immunocytochemistry platforms:

  • Utilize robotic liquid handling systems for antibody incubations

  • Implement standardized staining protocols across multiple cell lines

  • Combine with high-content imaging for quantitative analysis of KCNS2 expression

• Tissue microarray analysis:

  • Screen KCNS2 expression across multiple tissue types simultaneously

  • Quantify expression patterns across disease progression stages

  • Correlate expression with clinical outcomes in patient samples

• Flow cytometry applications:

  • Develop protocols for permeabilized cell detection of KCNS2

  • Use fluorescently-conjugated secondary antibodies for multi-parameter analysis

  • Combine with electrophysiological markers for functional correlation

These applications require careful validation of antibody specificity at the working dilutions used in automated systems (typically 1:100-1:500) and standardization of detection thresholds across batches . When implementing high-throughput approaches, researchers should include appropriate controls on each plate or array to account for potential batch effects.

What methodological advances are improving spatial resolution in KCNS2 antibody-based imaging?

Advanced imaging techniques for KCNS2 localization include:

• Super-resolution microscopy:

  • STORM/PALM approaches achieve 10-20 nm resolution of KCNS2 localization

  • SIM provides 100 nm resolution with standard immunofluorescence protocols

  • These techniques reveal nanoscale distribution of KCNS2 relative to other channel subunits

• Expansion microscopy:

  • Physical expansion of specimens allows visualization of subcellular localization

  • Compatible with standard KCNS2 antibodies at 1:100-1:200 dilutions

  • Particularly useful for studying subunit clustering in neuronal compartments

• Array tomography:

  • Serial ultrathin sections provide 3D reconstruction of KCNS2 distribution

  • Allows correlation of channel localization with ultrastructural features

  • Can be combined with multiple antibody labeling for contextual analysis

• CLEM (Correlative Light and Electron Microscopy):

  • Correlate KCNS2 immunofluorescence with electron microscopy ultrastructure

  • Requires specialized sample preparation and gold-conjugated secondary antibodies

  • Provides definitive localization at membrane/organelle interfaces

These advanced imaging approaches have revealed precise subcellular localization patterns of KCNS2, particularly in relation to other Kv channel subunits in neuronal and cardiac tissues . For optimal results with super-resolution techniques, higher primary antibody concentrations (1:50-1:100) are often required compared to conventional microscopy.