KCNC3 Antibody, FITC conjugated

What is the molecular target of KCNC3 antibody and what cellular functions does it regulate?

KCNC3 (also known as Kv3.3) is a voltage-gated potassium channel belonging to the Shaw-related subfamily that plays crucial roles in neuronal function. This channel is particularly important for:

• Rapid repolarization of fast-firing brain neurons

• Regulation of frequency, shape, and duration of action potentials in Purkinje cells

• Normal survival of cerebellar neurons through regulation of cellular Ca²⁺ homeostasis

• Normal motor function maintenance

• Reorganization of the cortical actin cytoskeleton and formation of actin veil structures in neuronal growth cones through interaction with HAX1 and the Arp2/3 complex

The FITC-conjugated antibodies targeting KCNC3 enable visualization of this channel's expression and localization in various experimental contexts.

How specific is the KCNC3 antibody, and what epitopes does it recognize?

The commercially available KCNC3 antibodies target different regions of the protein. The FITC-conjugated polyclonal antibody recognized in the search results targets:

• Amino acids 638-745 of the human KCNC3 protein

• The C-terminal region of KCNC3

Other available KCNC3 antibodies may target different epitopes, such as:

• Amino acids 701-718 of rat KCNC3 (intracellular, C-terminus)

• Amino acids 317-328

• Amino acids 468-517

• The middle region of the protein

When selecting an antibody, researchers should consider which epitope is most appropriate for their experimental question, particularly regarding accessibility in their experimental system.

What validation data supports the specificity of KCNC3 antibodies?

Validation of KCNC3 antibodies typically includes:

• Western blot analysis of rat brain membranes showing specific bands corresponding to KCNC3

• Testing in Kv3.3 knockout mice to confirm specificity

• Preincubation with blocking peptides to demonstrate epitope-specific binding

• Cross-reactivity testing across species (human, mouse, rat, and sometimes other mammals)

Researchers should request validation data from manufacturers or examine published literature using these antibodies in tissues relevant to their study.

What are the optimal protocols for immunofluorescence using FITC-conjugated KCNC3 antibodies?

Based on protocols used with KCNC3 antibodies in the literature, recommended parameters for immunofluorescence include:

• Sample preparation:

  • For cell culture: Fix cells with 4% formaldehyde for 20 minutes

  • Permeabilize with 0.1% Triton X-100 for 10 minutes

  • Block with 10% BSA

• Antibody application:

  • Dilution range: 1:100-1:500 for ELISA applications

  • For immunofluorescence, overnight incubation at 4°C is typically recommended

• Visualization considerations:

  • FITC excitation maximum: ~495 nm

  • FITC emission maximum: ~519 nm

  • When designing multi-channel imaging experiments, consider spectral overlap with other fluorophores

For specific tissues like brain sections or spiral ganglia neurons, optimization of fixation conditions may be required.

How should researchers optimize western blotting protocols for KCNC3 detection?

Optimal western blotting with KCNC3 antibodies requires attention to:

• Sample preparation:

  • Brain membranes or cellular fractions containing membrane proteins

  • Use of appropriate detergents for solubilization

• Gel electrophoresis considerations:

  • Expected molecular weight of KCNC3: ~80-85 kDa

  • Post-translational modifications, particularly glycosylation, may affect migration patterns

• Transfer and detection:

  • For FITC-conjugated antibodies used as primary antibodies, direct fluorescence scanning can be performed

  • Typical working dilution: 1:200 for western blotting applications

  • Include positive controls (brain tissue) and negative controls (tissue known not to express KCNC3)

• Result interpretation:

  • Wild-type KCNC3 and mutant forms may show different migration patterns due to altered post-translational modifications

How can researchers address non-specific binding when using KCNC3 antibodies?

Non-specific binding can be minimized through several approaches:

• Optimization of blocking conditions:

  • Increase BSA concentration in blocking buffer (up to 10%)

  • Consider alternative blocking agents like normal serum from the same species as the secondary antibody

• Antibody validation:

  • Always include a negative control using the KCNC3 blocking peptide (e.g., BLP-PC102)

  • Include tissue controls where KCNC3 is known to be absent

• Dilution optimization:

  • Titrate antibody concentrations to identify the optimal signal-to-noise ratio

  • For FITC-conjugated antibodies, excessive concentration can lead to high background fluorescence

• Cross-adsorption:

  • If cross-reactivity with related potassium channels is suspected, pre-adsorption against recombinant proteins of other Kv3 family members may improve specificity

What factors affect KCNC3 antibody detection in different subcellular compartments?

When investigating KCNC3 localization, researchers should consider:

• Fixation effects:

  • Over-fixation may mask epitopes, particularly in membrane proteins

  • Different fixatives (paraformaldehyde vs. methanol) may reveal different subcellular pools of KCNC3

• Trafficking considerations:

  • Wild-type KCNC3 localizes primarily to the plasma membrane

  • Mutant forms (e.g., R423H) show aberrant trafficking with strong perinuclear staining

  • Post-translational modifications affect trafficking - glycosylation patterns differ between wild-type and mutant KCNC3

• Detection sensitivity:

  • Expression levels vary across cell types and developmental stages

  • CHO cells have no detectable endogenous KCNC3 and can be used as negative controls or expression systems

How can KCNC3 antibodies be applied to investigate channel mutations associated with spinocerebellar ataxia?

KCNC3 mutations cause spinocerebellar ataxia type 13 (SCA13), and antibody-based approaches can help understand disease mechanisms:

• Comparative localization studies:

  • Wild-type KCNC3 shows normal plasma membrane localization

  • Mutant forms (R423H) demonstrate aberrant trafficking with perinuclear retention

  • Co-localization with organelle markers can identify the specific compartments where mutant proteins accumulate

• Protein interaction studies:

  • Co-immunoprecipitation using KCNC3 antibodies can identify novel interaction partners

  • Research has shown that KCNC3 interacts with EGFR (epidermal growth factor receptor), providing mechanistic insight into SCA13 pathology

• Functional expression systems:

  • CHO cells expressing fluorescently-tagged KCNC3 variants (wild-type vs. R423H, F448L, or R420H) can be used to study trafficking defects

  • Antibodies can validate expression levels when comparing electrophysiological data between constructs

What approaches can be used to study KCNC3 heteromerization with other Kv3-family channels?

KCNC3 may form heteromeric channels with other Kv3-family members, particularly Kv3.1. Research approaches include:

• Co-localization analysis:

  • Use differentially labeled antibodies against KCNC3 and Kv3.1 to examine overlapping expression patterns

  • Super-resolution microscopy can provide detailed information about channel clustering

• Co-immunoprecipitation:

  • KCNC3 antibodies can pull down channel complexes to identify interacting partners

  • Western blotting of immunoprecipitated material can reveal Kv3.1 association

• Functional evidence:

  • Knockout studies show that mice lacking both Kv3.1 and Kv3.3, but not either channel alone, display severe motor defects

  • This suggests functional redundancy or cooperation between these channels

• Dominant negative effects:

  • Mutant KCNC3 exerts dominant negative effects on wild-type protein

  • Similar mechanisms may apply to heteromeric channels containing KCNC3 and other Kv3 family members

How does KCNC3 expression change during neurodevelopment, and how can antibodies track these changes?

KCNC3 plays critical roles in neurodevelopment, with expression patterns changing throughout development:

• Developmental timeline:

  • SCA13 mutations in KCNC3 can cause congenital, non-progressive neurodevelopmental cerebellar hypoplasia

  • This suggests important roles for KCNC3 in early neuronal development and circuit formation

• Antibody applications:

  • Immunohistochemistry of brain sections at different developmental stages can map expression changes

  • Comparative studies between wild-type and KCNC3 mutant models can reveal developmental abnormalities

• Functional correlations:

  • Electrophysiological studies complemented by antibody localization can link channel expression to functional maturation of neuronal circuits

  • KCNC3 is particularly important in fast-firing neurons and contributes to high-frequency action potential generation

What is the relationship between KCNC3 and EGFR signaling in neurodevelopmental disorders?

Research has uncovered a surprising link between KCNC3 and EGFR signaling:

• Experimental evidence:

  • Co-expression of KCNC3 R423H with Drosophila EGFR (dEgfr) rescues eye phenotypes in Drosophila models

  • KCNC3 R423H expression in mammalian cells results in aberrant intracellular retention of human EGFR

• Mechanistic implications:

  • The neurodevelopmental consequences of KCNC3 R423H may be mediated through indirect effects on EGFR signaling in the developing cerebellum

  • This provides a potential mechanism for how ion channel mutations affect neurodevelopment beyond direct electrophysiological effects

• Research applications:

  • FITC-conjugated KCNC3 antibodies can be used for co-localization studies with EGFR

  • Time-course studies during development could reveal temporal relationships between KCNC3 and EGFR trafficking