KCNB1 Antibody, HRP conjugated

What is KCNB1 and how does it differ from KCNE1?

KCNB1 (also known as Kv2.1 or DRK1) is a voltage-gated potassium channel found in cardiomyocytes, skeletal muscles, vascular smooth muscles, placental vasculature, retina, and pancreatic beta-cells. It mediates voltage-dependent potassium ion permeability in excitable membranes and regulates neurotransmitter release, heart rate, insulin secretion, neuronal excitability, epithelial electrolyte transport, smooth muscle contraction, and cell volume . In contrast, KCNE1 functions as an ancillary protein that assembles as a beta subunit with voltage-gated potassium channel complexes, modulating gating kinetics and enhancing stability of channel complexes . KCNB1 forms the pore-forming alpha subunits of potassium channels, while KCNE1 serves as a regulatory subunit.

What are the molecular characteristics of KCNB1 antibodies?

KCNB1 antibodies are immunoglobulins specifically designed to recognize and bind to the KCNB1 protein. The monoclonal antibody clone L80/21 is raised in mice against a synthetic peptide corresponding to amino acids 837-853 (HMLPGGGAHGSTRDQSI) in the cytoplasmic C-terminus of rat Kv2.1 . This region shows 100% identity with mouse KCNB1 and 88% identity with the human ortholog . HRP-conjugated antibodies have horseradish peroxidase enzyme directly attached to the antibody molecule, enabling direct visualization in enzyme-based detection systems without requiring secondary antibodies.

What is the significance of HRP conjugation in KCNB1 antibodies?

HRP (horseradish peroxidase) conjugation provides several advantages in research applications. The enzyme catalyzes reactions with substrates to produce colorimetric, chemiluminescent, or fluorescent signals, allowing direct detection without secondary antibodies. This reduces background noise, simplifies experimental protocols, and decreases the time needed for detection procedures. The conjugation provides enhanced sensitivity for detecting low-abundance KCNB1 proteins in tissues or cell samples, making it especially valuable for quantitative analyses such as ELISA and western blotting.

What are the validated applications for KCNB1 antibodies?

KCNB1 antibodies have been validated for multiple experimental applications:

• Western Blot (WB): Used to detect KCNB1 protein in tissue or cell lysates, with observed binding at approximately 96 kDa for the native protein .

• Immunohistochemistry (IHC): Both paraffin-embedded and formalin-fixed samples can be analyzed for KCNB1 localization .

• Immunofluorescence (IF): For cellular localization studies .

• ELISA: Particularly suitable for HRP-conjugated antibodies for quantitative protein detection .

How should samples be prepared for optimal KCNB1 detection?

For western blotting:

• Extract proteins using buffers containing protease inhibitors to prevent degradation.

• Include phosphatase inhibitors if phosphorylation states are relevant.

• Denature samples at appropriate temperatures (typically 95°C for 5 minutes).

• Load adequate protein amounts (typically 20-50 μg for tissue lysates).

For immunohistochemistry:

• Fix tissues appropriately (formalin fixation is common).

• Consider antigen retrieval methods to expose epitopes that may be masked during fixation.

• Block endogenous peroxidase activity if using HRP-conjugated antibodies.

• Use appropriate blocking solutions to minimize non-specific binding.

What controls should be included when using KCNB1 antibodies?

Rigorous experimental design requires multiple controls:

• Positive control: Tissue or cell type known to express KCNB1 (brain tissue is excellent for this purpose) .

• Negative control: Tissue known not to express KCNB1 or where expression has been knocked down.

• Primary antibody omission: To detect non-specific binding of secondary antibodies or detection reagents.

• Isotype control: Using an irrelevant antibody of the same isotype (IgG3 for the monoclonal mentioned) .

• Peptide competition/blocking: Pre-incubating the antibody with the immunizing peptide should eliminate specific signal.

How can KCNB1 antibodies help investigate potassium channel complexes?

KCNB1 antibodies are valuable tools for studying channel complex formation through:

• Co-immunoprecipitation: Using KCNB1 antibodies to pull down the protein complex followed by detection of interacting partners. This approach has revealed interactions with various regulatory subunits.

• Proximity ligation assays: To visualize protein-protein interactions in situ.

• Immunofluorescence co-localization: To determine spatial relationships between KCNB1 and other channel components.

• Blue native PAGE: For studying intact channel complexes.

These techniques have helped elucidate how KCNB1 interacts with accessory subunits like KCNE proteins, which modulate channel function and are essential for understanding the regulation of potassium currents in various tissues .

How can KCNB1 antibodies contribute to neurological disorder research?

KCNB1 variants have been associated with epilepsy and neurodevelopmental disorders . Antibodies can help researchers:

• Analyze protein expression levels in patient-derived samples.

• Investigate subcellular localization changes associated with pathogenic variants.

• Study effects of mutations on channel complex formation.

• Correlate functional defects observed electrophysiologically with protein expression patterns.

A specific example is the characterization of KCNB1 variant c.595A>T (p.Ile199Phe) associated with epilepsy and centrotemporal spikes, where researchers used biochemical and electrophysiological approaches to determine that the variant resulted in partial loss of function .

What methodological approaches can distinguish between KCNB1 and related potassium channels?

What strategies can address non-specific binding in KCNB1 antibody applications?

Non-specific binding can significantly impact experimental results. Researchers should:

• Optimize blocking protocols: Use 3-5% BSA or 5% non-fat milk in TBS-T for western blots, and appropriate blocking solutions for immunohistochemistry.

• Titrate antibody concentrations: Test a range of dilutions to identify optimal signal-to-noise ratios.

• Adjust incubation conditions: Varying temperature (4°C overnight vs. room temperature) and duration can improve specificity.

• Increase washing stringency: More frequent and longer washes with appropriate buffers can reduce background.

• Add competing proteins: Adding irrelevant proteins (like BSA) to antibody dilutions can reduce non-specific interactions.

For HRP-conjugated antibodies specifically, endogenous peroxidase activity must be quenched in tissue samples using hydrogen peroxide treatment before antibody application.

How should researchers interpret contradictory data between KCNB1 protein detection and functional studies?

Discrepancies between immunodetection and functional data may arise for several reasons:

• Post-translational modifications: KCNB1 function can be regulated by phosphorylation, which may not affect antibody binding but alters channel function.

• Channel assembly issues: KCNB1 may be detected but not properly assembled into functional tetramers.

• Trafficking defects: Protein may be expressed but not correctly localized to the membrane.

• Regulatory subunit interactions: Presence or absence of partners like KCNE1 can dramatically alter function without changing detectable KCNB1 levels .

When facing contradictory data, researchers should:

• Employ multiple detection methods targeting different epitopes

• Verify subcellular localization through fractionation or imaging

• Assess the presence of known regulatory partners

• Consider functional heterogeneity in different cell populations within the sample

How are KCNB1 antibodies being used in cardiovascular and nervous system disease research?

KCNB1 antibodies are increasingly valuable in studying disease mechanisms:

• Cardiovascular diseases: KCNB1 has been implicated in over 18 publications related to cardiovascular diseases and 14 publications on heart diseases . Antibodies help researchers investigate altered channel expression in conditions like hypertension and congenital heart defects.

• Neurological disorders: With 9 publications connecting KCNB1 to nervous system diseases and 7 to brain diseases , antibodies are crucial for studying how channel dysfunction contributes to pathology. The discovery that KCNB1 variants cause early-infantile epileptic encephalopathy-26 has sparked extensive research using antibodies to analyze expression and localization patterns .

• Combined methodological approaches: Researchers are increasingly combining immunodetection with electrophysiology, genetics, and imaging to build comprehensive models of disease mechanisms.

What new methodological advances are improving KCNB1 research?

Recent technological developments enhancing KCNB1 research include:

• Super-resolution microscopy techniques that allow visualization of channel clustering and nanodomain organization beyond the diffraction limit.

• Proximity labeling approaches (BioID, APEX) that identify proximal proteins in the native cellular environment.

• Antibody-based proteomics combining immunoprecipitation with mass spectrometry to identify novel interaction partners.

• CRISPR-based genetic models that allow precise modeling of patient mutations and validation of antibody specificity.

• Patient-derived induced pluripotent stem cells differentiated into neurons or cardiomyocytes, providing human-relevant systems for studying KCNB1 pathophysiology.