KCNE3 Antibody, Biotin conjugated

What is KCNE3 and why is it significant in ion channel research?

KCNE3 (also known as MiRP2, HOKPP, or HYPP) functions as an ancillary protein that assembles as a beta subunit with voltage-gated potassium channel complexes composed of pore-forming alpha subunits. Its primary function is to modulate the gating kinetics and enhance stability of the channel complex . KCNE3 plays critical roles in multiple physiological processes through its interactions with different channel partners. When assembled with KCNB1, it modulates the gating characteristics of the delayed rectifier voltage-dependent potassium channel . When associated with KCNC4/Kv3.4, it helps form the subthreshold voltage-gated potassium channel in skeletal muscle that establishes the resting membrane potential in muscle cells . Additionally, when associated with KCNQ1/KCLQT1, it may form the intestinal cAMP-stimulated potassium channel involved in chloride secretion with nearly instantaneous activation and linear current-voltage relationship . Understanding KCNE3's modulatory effects on potassium channels provides valuable insights into electrophysiological processes and potential therapeutic targets.

What applications is the biotin-conjugated KCNE3 antibody suitable for?

The biotin-conjugated KCNE3 polyclonal antibody has been validated for multiple experimental applications that facilitate various research approaches:

This versatility allows researchers to employ the same antibody across multiple experimental platforms, ensuring consistency in target recognition while adapting to different research questions .

What is the specificity profile of this antibody?

This KCNE3 polyclonal antibody was developed using a KLH-conjugated synthetic peptide derived specifically from human KCNE3, corresponding to amino acids 51-103 of the 103-amino acid protein . The antibody demonstrates predicted cross-reactivity with KCNE3 from multiple species including human, mouse, rat, dog, cow, pig, horse, and chicken, making it suitable for comparative studies across different model organisms . This broad species reactivity is particularly valuable for evolutionary and comparative physiology studies examining conservation of KCNE3 structure and function.

How should I optimize Western blot protocols when using biotin-conjugated KCNE3 antibody?

When using biotin-conjugated KCNE3 antibody for Western blot analysis, several optimization strategies can enhance specificity and sensitivity:

• Sample preparation: KCNE3 is a membrane protein (103 amino acids) that requires proper solubilization. Use buffers containing 0.5% Igepal or similar mild detergents with protease inhibitor cocktails to maintain protein integrity .

• Blotting conditions: For membrane proteins like KCNE3, transfer at lower voltage (30V) overnight at 4°C in transfer buffer improves efficiency for transmembrane proteins .

• Blocking: Use 'SuperBlock' buffer or similar high-quality blocking solutions to minimize background without compromising specific signal .

• Detection strategy: Since the antibody is biotin-conjugated, avoid streptavidin-biotin detection systems that would create high background. Instead, use detection methods specific to rabbit IgG (the host species) .

• Dilution optimization: Begin with 1:1000 dilution and adjust based on signal-to-noise ratio for your specific experimental system .

Remember that KCNE3 may show multiple bands depending on post-translational modifications and sample preparation conditions. Appropriate positive and negative controls are essential for accurate interpretation.

What are the recommended procedures for co-immunoprecipitation studies involving KCNE3?

Co-immunoprecipitation (co-IP) studies with KCNE3 require careful consideration of protein-protein interaction preservation:

• Sample preparation: Homogenize tissue samples (e.g., brain) in appropriate buffer (50 mM Tris-Cl, 20 mM MgCl₂, 150 mM NaCl, 0.5% Igepal with protease inhibitor cocktail, pH 7) at 4°C .

• Antibody coupling: Covalently conjugate anti-KCNE3 antibody to protein A/G sepharose beads using coupling kits (e.g., ProFound kit) .

• Immunoprecipitation: Incubate lysate with antibody-conjugated beads overnight at 4°C, followed by thorough washing to remove non-specific proteins .

• Native conditions: For preserving protein complexes, consider using native conditions as described in the literature. This involves digitonin (1%) for solubilization and native electrophoresis buffers to maintain protein-protein interactions .

• Controls: Include negative controls such as antibodies against unrelated proteins (e.g., Kir2.1) to demonstrate specificity of interactions .

When investigating KCNE3 interactions with potassium channels like Kv12.2, two-step co-immunoprecipitation assays under native conditions can reveal whether multiple auxiliary subunits simultaneously interact with individual channel complexes .

How can I accurately quantify changes in KCNE3 membrane expression?

Quantifying membrane expression of KCNE3 requires techniques that specifically isolate plasma membrane fractions:

• Cell surface biotinylation: Use membrane-impermeable biotinylation reagents to label surface proteins, followed by NeutrAvidin capture and specific antibody detection .

• Fractionation protocols: Sequential centrifugation steps (10,000g followed by 100,000g) can separate membrane fractions prior to analysis .

• Verification of fraction purity: Include markers for specific cellular compartments (e.g., calnexin for ER) to verify the purity of your membrane fraction and avoid misinterpretation due to contamination .

• Quantification: Normalize membrane KCNE3 expression to total KCNE3 expression and use appropriate loading controls for each fraction .

• Statistical analysis: For accurate interpretation, perform experiments in triplicate minimum and apply appropriate statistical tests to determine significance of observed changes.

In published studies, these approaches have successfully demonstrated regulation of surface expression of channels like Kv12.2 by KCNE3, with changes in surface expression correlating well with electrophysiological data .

What controls are essential when working with KCNE3 antibody in immunoprecipitation experiments?

Robust controls are critical for interpreting KCNE3 immunoprecipitation data correctly:

• Input control: Always analyze a portion of the starting material alongside immunoprecipitated samples to verify protein presence in the original lysate .

• Negative antibody control: Include immunoprecipitation with an antibody targeting an unrelated protein (e.g., Kir2.1) to identify non-specific binding .

• Isotype control: Use rabbit IgG at the same concentration as the specific antibody to control for non-specific binding to the antibody class rather than the antigen .

• Blocking peptide control: Pre-incubate the antibody with the immunogenic peptide used to generate it (amino acids 51-103 of human KCNE3) to demonstrate binding specificity .

• Knockdown/knockout control: Where available, use KCNE3 siRNA knockdown or KCNE3 knockout models to confirm antibody specificity .

These controls help distinguish between true interactions and experimental artifacts, particularly important when studying subtle regulatory interactions between KCNE3 and its channel partners.

How can I troubleshoot inconsistent staining patterns in immunohistochemistry?

Inconsistent immunohistochemistry results with biotin-conjugated KCNE3 antibody may stem from several sources:

• Fixation optimization: KCNE3 is a membrane protein with both intracellular and extracellular domains. Test different fixation protocols (4% PFA, methanol, or acetone) to determine optimal epitope preservation .

• Antigen retrieval: For paraffin sections (IHC-P), compare different antigen retrieval methods (citrate buffer pH 6.0, EDTA buffer pH 9.0, or enzymatic retrieval) to identify optimal conditions .

• Blocking endogenous biotin: Since the antibody is biotin-conjugated, endogenous biotin in tissues must be blocked using avidin/biotin blocking kits prior to antibody application .

• Antibody concentration: Titrate the antibody across the recommended range (1:100-500 for IHC-F and 1:200-400 for IHC-P) to determine optimal signal-to-noise ratio for your specific tissue .

• Detection system: For biotin-conjugated antibodies, use streptavidin-conjugated reporter molecules (HRP or fluorophores) with sensitivity appropriate for expected expression levels .

If membrane staining is weak, consider light fixation and shorter permeabilization times to preserve membrane epitopes. For tissues with high background, longer blocking times and more stringent washing conditions may improve results.

How do I interpret differences between protein expression and functional data for KCNE3?

Discrepancies between KCNE3 protein detection and functional outcomes are common challenges requiring careful analysis:

• Post-translational modifications: KCNE3 function may be regulated by phosphorylation or other modifications that affect function without changing total protein levels detectable by the antibody .

• Protein-protein interactions: KCNE3 exists in complexes with different alpha-subunits, and these interactions may mask antibody epitopes without affecting protein levels .

• Subcellular localization: Total KCNE3 protein levels may remain constant while membrane expression changes, affecting function. Compare total protein with membrane fraction analysis .

• Temporal dynamics: Protein expression and functional effects may have different time courses, requiring careful time-point analysis .

• Compensatory mechanisms: Knockdown of KCNE3 may trigger upregulation of other KCNE family members that partially compensate functionally while appearing as clean knockdown in protein assays .

When comparing biochemical and electrophysiological data, consider that functional assays like patch-clamp are often more sensitive to subtle changes in channel properties than Western blots or immunohistochemistry can detect.

How can I study KCNE3-mediated modulation of channel gating kinetics?

Investigating KCNE3's effects on channel gating requires specialized electrophysiological approaches combined with molecular interventions:

• Heterologous expression systems: Co-express KCNE3 with channel alpha-subunits (KCNB1, KCNC4, or KCNQ1) in Xenopus oocytes or mammalian cells for controlled analysis of biophysical parameters .

• siRNA knockdown approach: Use targeted siRNA to reduce endogenous KCNE3 expression while monitoring channel currents, as demonstrated in studies showing ~3-fold increase in Kv12.2 channel surface expression following KCNE3 knockdown .

• Mutational analysis: Generate point mutations or truncations in the KCNE3 sequence (particularly within the 51-103 amino acid region) to identify domains critical for channel interaction and modulation .

• Native tissue analysis: Compare channel kinetics in tissues from wild-type vs. KCNE3-deficient models to assess physiological relevance of in vitro findings .

• Pharmacological dissection: Use selective potassium channel modulators to distinguish KCNE3-dependent components of native currents.

These approaches have revealed that KCNE3 regulates not only channel kinetics but also surface expression of interacting alpha-subunits, providing a dual mechanism for modulating cellular excitability .

What approaches can distinguish between KCNE family members in experimental systems?

Differentiating between KCNE3 and related family members (KCNE1-5) requires specialized techniques:

• Antibody specificity: The biotin-conjugated KCNE3 antibody targeting amino acids 51-103 provides specificity, as this region shows the highest sequence divergence among KCNE family members .

• Sequential immunoprecipitation: For complex samples, first immunoprecipitate with anti-KCNE3, then perform a second immunoprecipitation with antibodies against potential channel partners to isolate specific complexes .

• Native gel electrophoresis: Native PAGE (4-16% gradient gels) can separate intact channel complexes containing different KCNE subunits based on size and charge differences .

• Functional fingerprinting: Each KCNE imparts characteristic gating properties to partner channels. For example, KCNE3 produces nearly instantaneous activation with linear current-voltage relationships when co-expressed with KCNQ1, distinct from effects of other KCNE proteins .

• Co-expression studies: Selective knockdown or overexpression of individual KCNE members can help determine their relative contributions to observed channel properties .

Using these approaches in combination provides the most reliable differentiation between functionally similar but molecularly distinct KCNE family members.

What is the significance of KCNE3's simultaneous interaction with multiple channel types?

KCNE3's ability to interact with multiple potassium channel alpha-subunits has important physiological and pathophysiological implications:

• Tissue-specific regulation: KCNE3 interactions with different channel partners may vary by tissue, allowing cell-specific tuning of excitability properties .

• Pathophysiological relevance: Mutations in KCNE3 are associated with periodic paralysis (HOKPP/HYPP), suggesting its critical role in muscle excitability via regulation of specific channels .

• Competitive interactions: In tissues expressing multiple potential alpha-subunit partners, KCNE3 may preferentially associate with certain channels based on expression levels, subcellular localization, or regulatory signals .

• Heteromeric complexes: Native co-immunoprecipitation experiments suggest KCNE1 and KCNE3 may simultaneously interact with individual tetrameric Kv12.2 channels, creating complex regulatory possibilities .

• Therapeutic targeting: Understanding the specificity determinants for KCNE3 interactions could enable development of drugs that selectively modulate specific channel complexes without affecting others.

Research using native protein conditions has demonstrated that KCNE3 exists in complexes of approximately 500 kDa, consistent with association with tetrameric channel structures plus additional regulatory proteins .

What emerging technologies might enhance KCNE3 research?

Several cutting-edge approaches are positioned to advance our understanding of KCNE3 biology:

• Super-resolution microscopy: Techniques like STORM or PALM could reveal the nanoscale organization of KCNE3-containing channel complexes in the plasma membrane at unprecedented resolution.

• Proximity labeling approaches: BioID or APEX2 fusion proteins could identify the full complement of KCNE3-interacting proteins in living cells, beyond those detectable by traditional co-immunoprecipitation .

• Cryo-EM structural analysis: Capturing the structure of KCNE3 in complex with its channel partners would provide molecular insights into how this small protein exerts significant effects on channel function.

• Optogenetic control: Light-controlled KCNE3 expression or conformation changes could enable temporal precision in studying its regulatory effects on channel function.

• Patient-derived models: iPSC-derived excitable cells from patients with KCNE3 mutations could provide physiologically relevant systems for studying disease mechanisms and testing potential therapeutics.

These approaches would complement and extend the biochemical and electrophysiological techniques currently used to study KCNE3, potentially revealing new functions or regulatory mechanisms.

How can researchers effectively combine biochemical and electrophysiological approaches to study KCNE3?

Integrating multiple methodologies provides the most comprehensive understanding of KCNE3 function:

• Parallel analysis: Design experiments to measure both membrane expression (biotinylation assays) and channel function (patch-clamp) in the same experimental system following KCNE3 manipulations .

• Sequential immunoprecipitation with functional studies: Identify specific channel complexes by biochemical means, then express equivalent combinations in heterologous systems for functional characterization .

• Structure-function correlations: Combine mutational analysis with both binding studies and functional measurements to link specific protein domains to both physical interactions and functional effects .

• Temporal coordination: Include multiple time points in analyses to distinguish between acute vs. chronic effects of KCNE3 on channel properties and expression.

• Mathematical modeling: Develop quantitative models that integrate biochemical and biophysical data to predict how changes in KCNE3 expression or function affect cellular excitability.

This integrated approach has successfully demonstrated that KCNE3 regulation of Kv12.2 channels involves both direct modulation of gating properties and control of channel surface expression, mechanisms that may apply to other KCNE3-regulated channels .