KCNIP4 Antibody

What is KCNIP4 and why is it important in neurological research?

KCNIP4 (Kv channel interacting protein 4) is a member of the voltage-gated potassium (Kv) channel-interacting protein family belonging to the recoverin branch of the EF-hand superfamily. These proteins function as regulatory subunits of Kv4/D (Shal)-type voltage-gated rapidly inactivating A-type potassium channels . KCNIP4 is predominantly expressed in the brain and heart, where it contributes to the maintenance of neuronal and cardiomyocyte excitability . Its importance stems from its role in modulating channel density, inactivation kinetics, and recovery rate from inactivation in a calcium-dependent and isoform-specific manner . Research has implicated KCNIP4 in neurological disorders, making it a valuable target for studies on neural excitability and potential therapeutic interventions.

What types of KCNIP4 antibodies are available for research applications?

Several types of KCNIP4 antibodies are currently available for research:

When selecting an antibody, consider the specific research application, target species, and epitope of interest to ensure optimal experimental results.

How should KCNIP4 antibodies be stored and handled to maintain their efficacy?

For optimal storage and handling of KCNIP4 antibodies:

• Store at -20°C in the recommended buffer (typically PBS with 0.02% sodium azide and 50% glycerol at pH 7.3) .

• Antibodies remain stable for one year after shipment when properly stored .

• Aliquoting is generally unnecessary for -20°C storage, though smaller (20μl) sizes may contain 0.1% BSA .

• For lyophilized antibodies, reconstitute in distilled water to a final concentration of 1 mg/mL .

• Avoid multiple freeze-thaw cycles to preserve antibody activity and specificity .

• Once thawed for use, keep antibodies on ice during experiments to maintain binding properties.

Proper storage conditions ensure antibody stability and consistent experimental results.

What are the recommended dilutions for different experimental applications of KCNIP4 antibodies?

Optimal dilutions vary by application and specific antibody formulation:

It is strongly recommended to titrate each antibody in your specific testing system to determine optimal dilutions for your experimental conditions . Different tissue types and fixation methods may require adjustment of these dilution parameters.

How can researchers validate the specificity of KCNIP4 antibodies for their experimental model?

To validate KCNIP4 antibody specificity:

• Positive controls: Use tissues known to express KCNIP4 (brain tissues from human, rat, mouse, or pig) . Commercial antibodies have been validated in brain tissues where KCNIP4 is predominantly expressed.

• Western blot validation: Confirm a single band at the expected molecular weight (~29 kDa) . Multiple bands may indicate antibody cross-reactivity or alternative protein isoforms.

• Knockout/knockdown controls: Use KCNIP4 knockout tissues/cells or siRNA-mediated knockdown samples as negative controls.

• Peptide competition assays: Pre-incubate antibody with the immunizing peptide to confirm signal specificity.

• Antibody panel approach: Use multiple antibodies targeting different epitopes of KCNIP4 to confirm consistent localization and expression patterns.

• Correlation with mRNA expression: Verify that antibody signal correlates with KCNIP4 mRNA expression in various tissues.

This multi-faceted validation approach ensures reliable experimental outcomes and reduces the risk of misinterpreting results due to non-specific binding.

What are the best methodologies for detecting KCNIP4 isoforms resulting from alternative splicing?

KCNIP4 undergoes alternative splicing, producing multiple isoforms (variants) with different functional properties. For optimal detection of these isoforms:

• Isoform-specific antibodies: Use antibodies targeting unique N-terminal regions of specific variants. For example, antibodies recognizing the N-terminus of KCNIP4 Var I (KChIP4 N-14) versus those detecting the alternative Var IV (α-KCNIP4 Var IV) .

• Western blot analysis: Detect distinct KCNIP4 variants based on their molecular weights. Variant I appears at ~28 kDa while Variant IV appears at ~25 kDa .

• RT-PCR with isoform-specific primers: Design primers spanning exon junctions specific to each variant to quantify transcript levels.

• RNA sequencing: Employ RNA-seq to comprehensively identify and quantify all expressed KCNIP4 splice variants.

• Combined approaches: Use immunoprecipitation followed by mass spectrometry to confirm the identity of specific protein isoforms detected by antibodies.

• Splicing modulators: Use tools like the 38A ncRNA, which has been shown to drive alternative splicing of KCNIP4, as experimental controls for variant switching .

These approaches allow researchers to distinguish between functionally different KCNIP4 protein isoforms, which is critical given their distinct interactions with other proteins such as presenilins .

How can KCNIP4 protein-protein interactions be effectively studied using antibody-based techniques?

To study KCNIP4 protein-protein interactions:

• Co-immunoprecipitation (Co-IP):

  • Immunoprecipitate KCNIP4 using specific antibodies (e.g., KChIP4 L-14 IgGs) and analyze interacting partners by Western blotting .

  • This approach has successfully demonstrated KCNIP4-presenilin 2 interactions.

• Proximity Ligation Assay (PLA):

  • Detect protein interactions in situ with high sensitivity using antibodies against KCNIP4 and potential binding partners.

  • Provides spatial information about interaction sites within cells.

• Immunofluorescence co-localization:

  • Use dual immunofluorescence with antibodies against KCNIP4 and potential interacting proteins.

  • Different KCNIP4 variants show distinct co-localization patterns; Var I co-localizes with PS2 while Var IV does not .

• Pull-down assays:

  • Use recombinant KCNIP4 or peptide fragments to capture interacting proteins from cell lysates.

  • Follow with antibody detection to identify binding partners.

• Cross-linking coupled with immunoprecipitation:

  • Stabilize transient interactions before immunoprecipitation with KCNIP4 antibodies.

  • Analyze by mass spectrometry for comprehensive interactome profiling.

• Controls for specificity:

  • Use RNA polymerase III inhibitors (e.g., ML-60218) to modulate KCNIP4 variant expression and confirm interaction specificity .

  • Include negative controls such as PS1, which does not coimmunoprecipitate with KCNIP4 .

These methodologies have revealed that different KCNIP4 isoforms interact distinctly with other proteins, suggesting isoform-specific functions in cellular processes.

How can KCNIP4 antibodies be leveraged to study its role in neurological disorders?

KCNIP4 antibodies can be applied to neurological disorder research through several advanced approaches:

• Post-mortem tissue analysis:

  • Compare KCNIP4 expression patterns in brain tissue from patients with neurological disorders versus healthy controls using immunohistochemistry or immunofluorescence.

  • Antibodies validated for human brain tissue (particularly 60133-1-Ig and PACO36894) are suitable for this application .

• Alternative splicing dysregulation:

  • Investigate aberrant KCNIP4 splicing using isoform-specific antibodies in disease models.

  • The 38A-driven alternative splicing of KCNIP4 has implications for protein-protein interactions relevant to neurodegenerative conditions .

• Presenilin interaction studies:

  • Examine KCNIP4-presenilin interactions in Alzheimer's disease models using co-immunoprecipitation and co-localization studies.

  • KCNIP4 Var I interacts with PS2, while Var IV does not, suggesting implications for γ-secretase activity and APP processing .

• Electrophysiological correlates:

  • Combine antibody-based protein detection with electrophysiological recordings to correlate KCNIP4 expression with functional changes in neuronal excitability.

• Animal models of neurological disorders:

  • Analyze KCNIP4 expression in canine models of cerebellar ataxia, where KCNIP4 mutations have been identified .

  • Employ antibodies reactive with multiple species (e.g., 60133-1-Ig) for comparative studies.

• Therapeutic target validation:

  • Use antibodies to validate KCNIP4 as a potential therapeutic target by correlating its expression with disease progression and treatment response.

These applications can provide insights into the mechanistic role of KCNIP4 in neurological disorders and potentially identify novel therapeutic strategies.

What strategies can be employed when studying KCNIP4 in non-neuronal tissues where its expression is lower?

Detecting KCNIP4 in non-neuronal tissues with lower expression requires specialized approaches:

• Signal amplification techniques:

  • Employ tyramide signal amplification (TSA) for immunohistochemistry and immunofluorescence to enhance detection sensitivity.

  • Use highly sensitive chemiluminescent substrates for Western blotting.

• Protein concentration methods:

  • Perform immunoprecipitation to concentrate KCNIP4 before detection by Western blotting.

  • Use larger amounts of starting material from non-neuronal tissues.

• Optimized antibody selection:

  • Choose high-affinity antibodies with demonstrated sensitivity in Western blot applications (e.g., 13748-1-AP, dilution 1:1000-1:4000) .

  • Consider monoclonal antibodies for higher specificity in tissues with potential cross-reactive proteins.

• Subcellular fractionation:

  • Isolate membrane fractions to enrich for KCNIP4, which associates with membrane-bound potassium channels.

• Alternative detection methods:

  • Employ RNA analysis (qPCR, RNA-seq) to first confirm KCNIP4 expression at the transcript level.

  • Use mass spectrometry-based proteomics for unbiased protein detection.

• Positive controls and validation:

  • Always run parallel samples from brain tissue as positive controls.

  • Validate antibody specificity with recombinant KCNIP4 protein standards.

• Induction of expression:

  • Consider experimental conditions that might upregulate KCNIP4 in non-neuronal tissues before detection attempts.

These strategies can overcome the challenges of detecting low-abundance KCNIP4 in tissues beyond its primary sites of expression.

How can researchers effectively use KCNIP4 antibodies in acute protein degradation studies?

For KCNIP4 protein degradation studies using techniques like Trim-Away:

• Antibody selection criteria:

  • Choose antibodies with high affinity and specificity for KCNIP4.

  • Ensure antibodies recognize accessible epitopes in the native protein conformation.

  • Consider using multiple antibodies targeting different epitopes to confirm results.

• Trim-Away methodology adaptation:

  • Electroporate KCNIP4-specific antibodies into TRIM21-overexpressing cell lines to induce targeted protein degradation .

  • Establish optimal antibody concentrations (typically 0.5-2.0 μg per electroporation) based on KCNIP4 expression levels.

  • Monitor degradation kinetics through Western blotting at multiple time points (1-2 hours post-electroporation and up to 3-4 days) .

• Controls and validation:

  • Include isotype control antibodies to rule out non-specific effects.

  • Verify TRIM21 expression in your cell model before experiments.

  • Use siRNA-mediated KCNIP4 knockdown as a comparative approach.

• Functional readouts:

  • Combine degradation with electrophysiological recordings to assess functional consequences of acute KCNIP4 depletion.

  • Monitor calcium signaling as KCNIP4 functions as a calcium-binding protein.

• Degradation verification methods:

  • Confirm KCNIP4 degradation by Western blotting using antibodies recognizing different epitopes than those used for degradation.

  • Use immunofluorescence to visualize cellular depletion patterns.

This approach allows researchers to study the acute effects of KCNIP4 loss without genetic manipulation, providing insights into its immediate functional roles in cellular processes.

What are common challenges when detecting KCNIP4 isoforms by Western blot, and how can they be addressed?

Common challenges and solutions for KCNIP4 Western blotting:

• Multiple bands/isoform detection issues:

  • Challenge: KCNIP4 has multiple splice variants (approximately 5 isoforms) with similar molecular weights.

  • Solution: Use longer SDS-PAGE running times with gradient gels (10-15%) for better separation of closely migrating isoforms.

  • Solution: Employ isoform-specific antibodies that recognize unique N-terminal regions of specific variants .

• Low signal strength:

  • Challenge: KCNIP4 may be expressed at low levels in some tissues or cell types.

  • Solution: Increase protein loading (50-100 μg total protein) and optimize primary antibody concentration using a dilution series.

  • Solution: Use enhanced chemiluminescence detection systems or fluorescent Western blotting for improved sensitivity.

• Non-specific binding:

  • Challenge: Some antibodies may cross-react with other KCNIPs or EF-hand containing proteins.

  • Solution: Increase blocking time (1-2 hours) and use 5% non-fat dry milk in TBST as blocking buffer .

  • Solution: Increase washing duration and frequency (5-6 washes, 10 minutes each).

• Sample preparation issues:

  • Challenge: KCNIP4 degradation during extraction.

  • Solution: Include protease inhibitor cocktails in lysis buffers and keep samples cold throughout preparation.

  • Solution: Avoid harsh detergents that might denature the protein and affect antibody recognition.

• Antibody selection:

  • Challenge: Different antibodies detect different KCNIP4 epitopes with varying efficiency.

  • Solution: For general KCNIP4 detection, use antibodies recognizing conserved regions (e.g., 13748-1-AP) .

  • Solution: For isoform-specific detection, select antibodies raised against unique regions of each variant .

Researchers should validate their Western blotting protocol using positive control samples (e.g., brain tissue) where KCNIP4 is known to be expressed at detectable levels .

How can researchers distinguish between technical artifacts and true biological variation when interpreting KCNIP4 antibody results?

To distinguish artifacts from biological variation in KCNIP4 antibody studies:

• Multiple antibody approach:

  • Use at least two different KCNIP4 antibodies recognizing distinct epitopes.

  • True biological variation should be consistently detected across different antibodies.

  • Discrepancies between antibodies may indicate epitope masking or technical artifacts.

• Correlation with mRNA data:

  • Compare protein expression patterns with KCNIP4 mRNA levels by RT-qPCR or RNA-seq.

  • Discordance between protein and mRNA data requires further investigation.

• Biological replicates and statistical analysis:

  • Perform experiments with sufficient biological replicates (minimum n=3).

  • Apply appropriate statistical tests to determine if variations are significant.

  • Report both biological and technical variability in results.

• Positive and negative controls:

  • Include tissues/cells known to express (brain) or not express KCNIP4.

  • Use genetic manipulation (siRNA knockdown, CRISPR knockout) to validate antibody specificity.

• Blocking peptide competition:

  • Pre-incubate antibody with immunizing peptide to confirm signal specificity.

  • True KCNIP4 signal should be dramatically reduced or eliminated.

• Alternative detection methods:

  • Confirm key findings using non-antibody methods such as mass spectrometry.

  • For localization studies, validate with fluorescent protein tagging or in situ hybridization.

• Response to physiological stimuli:

  • Assess whether changes in KCNIP4 detection correspond to expected physiological responses.

  • For example, evaluate correlation with changes in Kv4 channel expression levels, which are coupled to KChIP levels .

What are the key considerations when comparing results from different KCNIP4 antibodies in the same experimental system?

When comparing results from different KCNIP4 antibodies:

• Epitope differences and accessibility:

  • Map the epitopes recognized by each antibody relative to KCNIP4's structure.

  • Antibodies recognizing the N-terminus (e.g., KChIP4 N-14) detect specific variants like Var I but not Var IV .

  • Antibodies targeting internal regions (e.g., KChIP4-L14) may detect multiple variants .

  • Consider epitope masking in protein complexes or due to post-translational modifications.

• Antibody format and performance characteristics:

  • Compare monoclonal (e.g., 60133-1-Ig) versus polyclonal (e.g., 13748-1-AP) antibodies .

  • Monoclonals offer higher specificity but may be more sensitive to epitope changes.

  • Polyclonals provide broader detection but potentially more background.

• Cross-reactivity profiles:

  • Assess potential cross-reactivity with other KCNIPs (KChIP1-3) which share significant homology.

  • Review validation data showing specificity testing against related proteins.

• Application optimization:

  • Different antibodies have distinct optimal dilutions for various applications (e.g., WB: 1:1000-1:4000; IHC: 1:50-1:500) .

  • Ensure each antibody is used under its optimal conditions before comparing results.

• Standardized controls:

  • Use identical positive and negative controls across all antibody tests.

  • Include recombinant KCNIP4 protein standards when possible.

• Normalization and quantification approaches:

  • Apply consistent normalization methods when quantifying signals from different antibodies.

  • Use total protein normalization rather than single housekeeping proteins when possible.

• Documentation of discrepancies:

  • Document and investigate discrepancies between antibodies rather than selecting only concordant results.

  • Discrepancies may reveal biologically meaningful information about protein isoforms or modifications.

These considerations ensure reliable interpretation when using multiple KCNIP4 antibodies to validate experimental findings.

How might KCNIP4 antibodies contribute to understanding the role of this protein in neurodegenerative diseases?

KCNIP4 antibodies offer significant potential for advancing neurodegenerative disease research:

• Alzheimer's disease mechanisms:

  • KCNIP4 interacts with presenilin 2 (PS2), a component of the γ-secretase complex involved in APP processing .

  • Antibodies can track KCNIP4 isoform switching (Var I to Var IV) which disrupts PS2 interaction, potentially affecting amyloid processing .

  • Combined with 38A ncRNA studies, antibodies can help elucidate how alternative splicing of KCNIP4 influences disease progression.

• Neuronal excitability and neurodegeneration:

  • KCNIP4 modulates Kv4 channels, affecting neuronal excitability .

  • Antibodies can map KCNIP4 expression changes in different brain regions during disease progression.

  • Correlative studies linking KCNIP4 expression patterns with electrophysiological changes could reveal mechanisms of neuronal dysfunction.

• Biomarker development:

  • Quantification of specific KCNIP4 isoforms in cerebrospinal fluid or blood using antibody-based assays may provide novel biomarkers.

  • Changes in KCNIP4-PS2 interaction could serve as an early marker for altered amyloid processing.

• Therapeutic target validation:

  • Antibodies can verify KCNIP4 as a therapeutic target by correlating its expression or interactions with disease severity.

  • Monitoring KCNIP4 expression in response to experimental treatments can help assess efficacy.

• Genetic models of neurodegeneration:

  • Antibodies can characterize KCNIP4 expression in animal models carrying mutations in related genes.

  • The link between KCNIP4 mutations and cerebellar ataxia in dogs provides a model for studying neurodegeneration .

• Cellular stress responses:

  • Track KCNIP4 expression during cellular stress conditions relevant to neurodegeneration using immunocytochemistry or Western blotting.

  • Investigate calcium-dependent regulation of KCNIP4 function in stress conditions.

These applications could reveal new pathways contributing to neurodegenerative processes and identify novel intervention points for therapeutic development.

What novel techniques are emerging for studying KCNIP4 protein dynamics and regulation with antibody-based approaches?

Emerging techniques for studying KCNIP4 using antibody-based approaches:

• Super-resolution microscopy:

  • Apply STORM or PALM imaging with fluorescently-labeled KCNIP4 antibodies to visualize nanoscale distribution at the plasma membrane and in relation to Kv4 channels.

  • Resolving protein clusters beyond the diffraction limit provides insights into functional organization.

• Live-cell antibody fragment imaging:

  • Use Fab fragments or nanobodies derived from KCNIP4 antibodies conjugated to fluorescent proteins.

  • Monitor real-time trafficking and dynamics of KCNIP4 in living neurons.

• Antibody-based proximity labeling:

  • Conjugate KCNIP4 antibodies with proximity labeling enzymes (APEX2, BioID, TurboID).

  • Map the local interactome of KCNIP4 in its native cellular context.

• Single-molecule pull-down (SiMPull):

  • Combine single-molecule fluorescence microscopy with antibody pull-down to analyze KCNIP4 complexes at the single-molecule level.

  • Determine stoichiometry and composition of native KCNIP4-containing complexes.

• Intrabodies for functional manipulation:

  • Develop intracellularly expressed antibody fragments (intrabodies) targeting specific KCNIP4 domains.

  • Disrupt specific protein-protein interactions without eliminating the entire protein.

• Optogenetic antibody control:

  • Engineer light-responsive antibody fragments that can be temporally controlled.

  • Allow precise temporal disruption of KCNIP4 function in specific subcellular compartments.

• Degradation technologies beyond Trim-Away:

  • Apply emerging technologies like dTAG or Turbodepletion to KCNIP4 for even more rapid protein degradation.

  • Couple with high-temporal resolution electrophysiology to capture acute effects of KCNIP4 loss.

• Spatially-resolved proteomics:

  • Combine antibody-based tissue clearing methods with mass spectrometry imaging.

  • Map KCNIP4 distribution across brain regions with corresponding interactors.

These emerging techniques promise to reveal new aspects of KCNIP4 biology beyond what conventional antibody applications have shown.

What are the most promising directions for developing more specific tools to study distinct KCNIP4 splice variants?

Promising directions for developing splice variant-specific tools:

• Epitope-targeted monoclonal antibody development:

  • Generate monoclonal antibodies against unique junction sequences created by alternative splicing.

  • Target the N-terminal regions that differ between variants (particularly between Var I and Var IV) .

  • Utilize phage display technology to select antibodies with extreme specificity for splice junctions.

• Recombinant antibody engineering:

  • Develop recombinant antibody fragments (scFv, Fab) with enhanced specificity for particular KCNIP4 variants.

  • Engineer affinity-matured antibodies through directed evolution approaches.

• Aptamer development:

  • Generate RNA or DNA aptamers specifically recognizing distinct KCNIP4 splice variants.

  • These can complement antibody-based approaches and offer advantages for certain applications.

• Splice-switching oligonucleotides (SSOs):

  • Design SSOs to selectively modulate KCNIP4 splicing toward specific variants.

  • Use these in combination with antibodies to validate isoform-specific detection.

• CRISPR-based isoform tagging:

  • Apply CRISPR/Cas9 genome editing to insert epitope tags or fluorescent proteins into endogenous KCNIP4 in an isoform-specific manner.

  • Create cellular models expressing tagged versions of specific variants for validation of antibodies.

• Isoform-specific degradation approaches:

  • Design degradation tags that can be inserted into specific KCNIP4 splice variants.

  • Develop antibodies conjugated to E3 ligase recruiting molecules for isoform-specific degradation.

• Computational epitope prediction and validation:

  • Use structural biology and computational approaches to predict optimal unique epitopes in each variant.

  • Validate predicted epitopes experimentally before large-scale antibody production.

• Mass spectrometry reference maps:

  • Develop detailed mass spectrometry fingerprints of each KCNIP4 isoform.

  • Use these as reference standards to validate antibody specificity.

These approaches would significantly advance our ability to study the diverse functions of KCNIP4 splice variants in normal physiology and disease states.