KCNB1 Antibody, Biotin conjugated

What is KCNB1 and what is its role in neuronal function?

KCNB1 encodes the KV2.1 voltage-gated potassium channel, which plays a crucial role in regulating neuronal excitability. Mutations in this gene can result in early-onset epileptic encephalopathy by causing loss of ion selectivity and gain of a depolarizing inward cation conductance . The protein has a calculated molecular weight of 96 kDa, though it typically appears at approximately 130 kDa in experimental conditions due to post-translational modifications .

What are the standard applications for KCNB1 antibodies in research?

KCNB1 antibodies are commonly employed in:

The antibody demonstrates reactivity with human and mouse samples, with cited reactivity in rat samples as well .

What tissue samples are optimal for studying KCNB1 expression?

Positive Western blot detection has been reported in neuroblastoma cell lines (SH-SY5Y), while positive IHC detection is documented in mouse brain tissue . For immunohistochemistry, antigen retrieval with TE buffer pH 9.0 is recommended, with citrate buffer pH 6.0 as an alternative method . These parameters are critical for accurate detection and quantification of KCNB1 in experimental settings.

How can researchers utilize cell surface biotinylation to study KCNB1 trafficking?

Cell surface biotinylation offers a powerful approach to investigating KCNB1 channel trafficking and membrane expression. The methodology involves:

• Surface protein labeling with biotin reagents (e.g., sulfo-NHS-SS-biotin)

• Precipitation with streptavidin

• Immunoblotting with KCNB1-specific antibodies

For studying internalization kinetics, researchers can implement glutathione-cleavable biotin in conjunction with timed glutathione stripping. This protocol allows quantification of internalized vs. surface-expressed KCNB1 channels . In oxidative stress studies, this technique has revealed that oxidative conditions may lead to accumulation of KCNB1 channels in the plasma membrane by impairing their endocytosis .

What experimental approaches can differentiate between wild-type and mutant KCNB1 proteins?

Differentiating wild-type from mutant KCNB1 proteins requires a multi-faceted approach:

• Electrophysiological analysis:

  • Whole-cell patch-clamp recordings under standardized conditions

  • Bath solution (in mmol/L): 138 NaCl, 5.4 KCl, 2 CaCl2, 1 MgCl2, 10 glucose, 10 HEPES (pH 7.4)

  • Pipette solution (in mmol/L): 140 KCl, 2 MgCl2, 10 EGTA, 10 HEPES, 5 Mg-ATP (pH 7.4)

• Expression configurations:

  • Homomeric: expressing solely wild-type or variant subunits

  • Heteromeric: co-expressing wild-type and variant subunits

• Functional classification system:

  • Partial vs. complete loss of function (based on current amplitude)

  • Dominant-negative vs. non-dominant-negative effects (based on interference with wild-type function)

• Protein expression analysis:

  • Western blotting for total protein levels

  • Surface biotinylation for membrane expression

What controls are necessary when investigating dominant-negative effects of KCNB1 variants?

When studying dominant-negative (DN) effects, where mutant subunits interfere with wild-type function, implement the following controls:

• Expression verification:

  • Equal expression of wild-type and mutant proteins via Western blotting

  • Testing multiple ratios of wild-type to mutant DNA for dose-dependency analysis

• Surface trafficking assessment:

  • Quantification of membrane vs. total protein expression

  • Comparison of trafficking efficiency between wild-type, mutant, and co-expressed conditions

• Functional controls:

  • Wild-type homomeric channels (positive control)

  • Mutant homomeric channels (intrinsic functionality assessment)

  • Non-transfected cells (negative control)

Recent research has demonstrated that certain KCNB1 variants display reduced membrane expression compared to wild-type, suggesting defective cell-surface trafficking .

How can researchers correlate KCNB1 variant functional effects with clinical phenotypes?

Establishing genotype-phenotype correlations for KCNB1 variants requires:

• Functional classification:

  • Categorize variants based on electrophysiological properties

  • Assess protein expression and trafficking patterns

  • Determine dominant-negative potential

• Clinical data collection:

  • Seizure characterization

  • EEG patterns

  • Developmental milestones

  • Neuroimaging findings

  • Response to therapeutic interventions

• Statistical correlation:

  • Group variants by functional effect

  • Compare clinical outcomes across functional categories

  • Identify predictive biomarkers of disease severity

This approach has recently been applied to correlate variant effects with clinical presentations in KCNB1-related neurodevelopmental disorders .

What methodologies are effective for studying KCNB1 in epileptic encephalopathy models?

For investigating KCNB1's role in epileptic encephalopathy:

• Genetic identification:

  • High-coverage whole exome sequencing (WES)

  • Lower-coverage whole genome sequencing (WGS)

  • Targeted sequencing of epilepsy gene panels

• Functional characterization:

  • Electrophysiological assessment of ion selectivity

  • Measurement of aberrant conductances

  • Protein expression and trafficking analysis

• In vitro modeling:

  • Transfection of neuronal or CHO-K1 cell lines

  • Site-directed mutagenesis to introduce patient-specific variants

  • Co-expression systems to model heterozygous mutations

Research has demonstrated that de novo KCNB1 mutations can result in loss of ion selectivity and gain of a depolarizing inward cation conductance, mechanistically explaining their pathogenicity in epileptic encephalopathy .

How can oxidative stress-induced modifications of KCNB1 be studied using biotinylated antibodies?

Oxidative stress induces KCNB1 channel modifications including oligomerization, which can be investigated through:

• Detection methods:

  • Non-reducing vs. reducing SDS-PAGE

  • Western blotting for high-molecular-weight complexes

  • FITC-conjugated biotin labeling to visualize protein distribution

• Internalization assays:

  • Glutathione-cleavable sulfo-NHS-SS-biotin labeling

  • Glutathione stripping to quantify internalization rates

  • Comparison between oxidized and non-oxidized conditions

• Cysteine mutant controls:

  • C73A KCNB1 mutants serve as important controls

  • Comparison of wild-type and mutant responses to oxidation

Experimental evidence indicates that oxidation leads to KCNB1 oligomerization and aggregation in glycolipid rafts, potentially triggering apoptotic pathways .

What are the key validation steps for KCNB1 antibodies in experimental protocols?

Thorough validation of KCNB1 antibodies requires:

• Specificity confirmation:

  • Knockout/knockdown controls (KD/KO experiments)

  • Western blot band detection at expected molecular weight (~130 kDa)

• Cross-reactivity testing:

  • Verification with human, mouse, and rat samples as appropriate

  • Testing in relevant cell lines (e.g., SH-SY5Y for KCNB1)

• Application-specific validation:

  • Western blot: Optimizing protein extraction and loading conditions

  • IHC: Testing different antigen retrieval methods (TE buffer pH 9.0 or citrate buffer pH 6.0)

  • Various dilutions to determine optimal signal-to-noise ratio

• Storage and handling:

  • Maintain at -20°C in appropriate buffer conditions

  • Follow stability guidelines for long-term storage

Proper validation ensures experimental reliability and reproducibility when studying KCNB1 in various research contexts.

How can KCNB1 antibodies contribute to understanding channel assembly and subunit interactions?

Investigating KCNB1 channel assembly requires:

• Co-immunoprecipitation studies:

  • Epitope-tagged wild-type and mutant subunits

  • Analysis of heteromeric vs. homomeric assembly

  • Quantification of subunit stoichiometry

• FRET/BRET techniques:

  • Fluorescently labeled channel subunits

  • Real-time monitoring of protein-protein interactions

  • Assessment of assembly dynamics in living cells

• Cross-linking approaches:

  • Chemical cross-linking of adjacent subunits

  • Mass spectrometry identification of interaction sites

  • Validation with site-directed mutagenesis

These methodologies provide crucial insights into how normal and mutant KCNB1 subunits assemble into functional tetrameric channels and how mutations might disrupt this process.

What considerations apply when studying KCNB1 in neuronal versus heterologous expression systems?

Key considerations include:

• Expression system differences:

  • Neuronal systems (primary cultures, neuroblastoma lines) provide physiological context

  • Heterologous systems (CHO-K1, HEK293) offer clean background but lack neuronal factors

• Experimental adjustments:

  • Neuronal systems: Account for endogenous KCNB1 expression

  • Heterologous systems: Consider effects of missing neuronal-specific interacting proteins

• Trafficking analysis:

  • Neuronal cells may exhibit different trafficking mechanisms

  • Heterologous systems may require co-expression of neuronal trafficking factors

• Functional testing:

  • Patch-clamp protocols may need adjustment between systems

  • Current density normalization essential for comparative analyses

Understanding these system-specific factors is critical for translating findings between experimental models and interpreting their relevance to human disease.