Phospho-KCNB1 (S805) Antibody

What is KCNB1 and why is its phosphorylation at S805 significant?

KCNB1, also known as Kv2.1, DRK1, or h-DRK1, belongs to the potassium channel family and B (Shab) subfamily. It functions as a voltage-gated potassium channel that mediates the voltage-dependent potassium ion permeability of excitable membranes. Channels open or close in response to voltage differences across the membrane, allowing potassium ions to pass according to their electrochemical gradient . Phosphorylation at serine 805 (S805) represents one of multiple phosphorylation sites that may regulate channel function, affecting neuronal excitability, action potential characteristics, and cellular responses to stimuli. This specific phosphorylation site serves as a potential biomarker for channel activity in various physiological and pathological conditions.

What specific applications is the Phospho-KCNB1 (S805) Antibody validated for?

The Phospho-KCNB1 (S805) Antibody has been primarily validated for Western Blot (WB) and ELISA applications . For Western blot applications, recommended dilutions typically range from 1:500 to 1:2000, while ELISA applications may use dilutions up to 1:40000 . When employing this antibody in Western blot protocols, researchers should optimize blocking conditions and incubation times to maximize signal-to-noise ratio, particularly when working with complex tissue samples like brain extracts where multiple potassium channel isoforms may be present.

How should researchers optimize storage conditions for maximum antibody stability?

For optimal stability, store the Phospho-KCNB1 (S805) Antibody at 4°C for short-term storage (1-2 weeks) . For long-term storage, aliquot the antibody to avoid repeated freeze-thaw cycles and store at -20°C . The antibody is typically supplied in a buffer containing PBS with 50% glycerol, 0.5% BSA, and 0.02% sodium azide at approximately pH 7.3 . When preparing working dilutions, it is advisable to use fresh buffer and maintain the antibody at 4°C rather than room temperature. Tracking antibody performance over time with consistent positive controls can help monitor potential degradation.

What are the recommended protocols for Western blot detection of phosphorylated KCNB1?

For optimal detection of phosphorylated KCNB1 at S805, researchers should follow these methodological guidelines:

• Sample preparation: Tissue or cell lysates should be prepared with phosphatase inhibitors (e.g., sodium fluoride, sodium orthovanadate) to preserve phosphorylation status.

• Gel selection: Use lower percentage (7-8%) SDS-PAGE gels to effectively resolve the approximately 100 kDa KCNB1 protein .

• Transfer conditions: Transfer proteins to PVDF membranes using standard wet transfer methods (25V overnight at 4°C is recommended for large proteins).

• Blocking: Block membranes with 5% BSA in TBST rather than milk, as milk contains phosphatases that may dephosphorylate your target.

• Antibody dilution: Dilute the Phospho-KCNB1 (S805) antibody in the range of 1:500 to 1:1000 in TBST with 5% BSA .

• Incubation: Incubate with primary antibody overnight at 4°C with gentle rocking.

• Detection: Use appropriate secondary antibodies (anti-rabbit IgG) conjugated to HRP or fluorescent tags, following the dilutions recommended by the manufacturer.

• Controls: Include a peptide competition assay with the phospho-specific peptide to confirm specificity, as demonstrated in previous studies .

How can researchers distinguish between total KCNB1 and phosphorylated KCNB1?

To effectively distinguish between total and phosphorylated KCNB1, implement a multi-pronged approach:

• Parallel detection: Run duplicate samples on separate blots or re-probe the same blot (after stripping) with both phospho-specific and total KCNB1 antibodies.

• Phosphatase treatment: Treat control samples with lambda phosphatase to remove phosphorylation and confirm the specificity of the phospho-antibody signal.

• Normalization strategy: Calculate the ratio of phospho-KCNB1 to total KCNB1 to account for variations in total protein expression between samples.

• Dephosphorylation controls: Include samples from treatments known to decrease S805 phosphorylation as biological controls.

• Peptide competition: Perform peptide competition assays with both phosphorylated and non-phosphorylated peptides to demonstrate phospho-specificity .

When interpreting results, be aware that changes in band intensity could reflect either altered phosphorylation status or changes in total protein levels, making the combined approach essential.

How can the Phospho-KCNB1 (S805) Antibody be utilized in studies of neurological disorders?

KCNB1 mutations are associated with developmental and epileptic encephalopathies (DEE) and other neurodevelopmental disorders (NDD) . Researchers investigating these conditions can employ the Phospho-KCNB1 (S805) Antibody in several sophisticated approaches:

• Patient-derived samples: Compare phosphorylation patterns in patient-derived neurons (from iPSCs) with those from healthy controls to investigate whether disease-associated mutations affect S805 phosphorylation.

• Animal models: Use the antibody in immunohistochemistry or Western blot analyses of brain tissues from animal models expressing KCNB1 mutations to determine regional and cell-type-specific alterations in phosphorylation.

• Electrophysiology correlation: Combine patch-clamp recordings with phospho-antibody staining to correlate channel function with phosphorylation status, particularly in studies examining how KCNB1 mutations affect channel kinetics .

• Drug response studies: Monitor changes in S805 phosphorylation in response to antiepileptic drugs to potentially identify novel therapeutic mechanisms or biomarkers of treatment response.

• Stress-response analysis: Investigate how cellular stressors alter KCNB1 phosphorylation, particularly relevant given findings that stress-induced translation of KCNB1 contributes to enhanced excitability of neurons .

What methodological approaches can detect changes in S805 phosphorylation in response to cellular stimuli?

To effectively monitor dynamic changes in S805 phosphorylation:

• Time-course experiments: Design protocols with multiple time points after stimulus application to capture the kinetics of phosphorylation and dephosphorylation.

• Pharmacological interventions: Apply specific kinase inhibitors or activators to identify the signaling pathways regulating S805 phosphorylation.

• Immunocytochemistry-flow cytometry: This combined approach allows quantification of phosphorylation levels in heterogeneous cell populations, as demonstrated in studies of KCNB1 variants .

• Quantitative Western blotting: Use a standard curve of recombinant phosphorylated protein for absolute quantification or normalize to total KCNB1 levels for relative quantification.

• Phospho-specific ELISA: Develop sandwich ELISA protocols using capture antibodies against total KCNB1 and detection with the phospho-specific antibody for high-throughput screening.

What are common challenges in detecting phosphorylated KCNB1 in native tissue samples?

Researchers frequently encounter these technical hurdles when working with endogenous phosphorylated KCNB1:

• Rapid dephosphorylation: Phosphorylation status can change rapidly during sample preparation. Address this by using ice-cold buffers containing phosphatase inhibitor cocktails and processing samples quickly.

• Low abundance: In some tissues, the phosphorylated form may represent only a small fraction of total KCNB1. Consider immunoprecipitation with total KCNB1 antibodies followed by Western blotting with the phospho-specific antibody to enrich for your target.

• Cross-reactivity: The antibody may detect other phosphorylated potassium channels due to sequence similarities. Validate specificity using KCNB1 knockout/knockdown controls or peptide competition assays .

• Regional heterogeneity: Phosphorylation levels may vary across brain regions or cell types. Use microdissection techniques or single-cell approaches when working with heterogeneous tissues.

• Activity-dependent phosphorylation: Channel phosphorylation may depend on neuronal activity states. Consider how experimental conditions (anesthesia, handling, time of day) might affect phosphorylation status.

How can researchers validate the specificity of the Phospho-KCNB1 (S805) Antibody?

A robust validation strategy includes:

• Peptide competition: Pre-incubate the antibody with the phosphorylated immunogen peptide versus non-phosphorylated peptide to demonstrate phospho-specificity .

• Phosphatase treatment: Treat samples with lambda phosphatase to remove phosphorylation and confirm signal reduction.

• Mutagenesis: Express S805A mutant KCNB1 (where serine is replaced with alanine to prevent phosphorylation) and confirm absence of antibody binding.

• Kinase manipulation: Use pharmacological or genetic approaches to modulate kinases predicted to target S805 and observe corresponding changes in antibody signal.

• Stimulus-response: Apply stimuli known to alter KCNB1 phosphorylation and confirm expected changes in signal.

• Multiple detection methods: Compare results across techniques (Western blot, immunocytochemistry, ELISA) to build confidence in antibody specificity.

What is known about the relationship between KCNB1 mutations and channel phosphorylation?

Studies of KCNB1 variants associated with epileptic encephalopathies have revealed complex relationships between mutations and channel function :

• Functional impact spectrum: Different mutations cause varying biophysical defects, including altered voltage-sensitivity, ion selectivity, and cell-surface expression . The relationship between these functional changes and S805 phosphorylation remains largely unexplored.

• Dominant-negative effects: Some KCNB1 variants (W370R, P385T, F416L) exhibit dominant-negative interactions with wild-type channels when co-expressed , potentially affecting the phosphorylation status of heteromeric channel complexes.

• Surface expression: Variants with reduced cell-surface expression may show altered patterns of phosphorylation due to differences in membrane localization or interactions with regulatory proteins .

• Regional vulnerability: Mutations may have differential effects on phosphorylation across brain regions, potentially explaining the specific neurological phenotypes associated with different variants.

• Signaling pathway integration: How KCNB1 mutations affect the channel's integration into neuronal signaling networks, including its regulation by phosphorylation, represents an important research frontier.

Methodologically, researchers investigating these relationships should consider combining electrophysiological recordings with phospho-specific antibody detection in heterologous expression systems and in patient-derived neurons.

What approaches can distinguish between the functional effects of phosphorylation at different sites on KCNB1?

KCNB1 contains multiple phosphorylation sites that may have distinct or interacting functional effects. To dissect site-specific contributions:

• Site-directed mutagenesis: Create single and combination phospho-mimetic (S→D or S→E) and phospho-resistant (S→A) mutations at key sites including S805.

• Phospho-specific antibody panels: Use antibodies targeting different phosphorylation sites to create a comprehensive phosphorylation profile.

• Mass spectrometry: Employ quantitative phosphoproteomics to simultaneously monitor multiple phosphorylation sites and their relative occupancy.

• Kinase/phosphatase manipulation: Use specific activators or inhibitors of kinases/phosphatases targeting different sites to dissect their unique contributions.

• Structure-function correlations: Correlate electrophysiological recordings with phosphorylation status at different sites to map functional consequences.

• Dynamic imaging: Use fluorescent biosensors sensitive to specific phosphorylation events to monitor site-specific phosphorylation in real-time.