KCNIP1 Antibody

What is KCNIP1 and why is it biologically significant?

KCNIP1 belongs to the neural calcium binding protein superfamily and contains EF-hand-like domains that respond to changes in intracellular calcium levels . Its biological significance spans multiple functions:

• It serves as an integral subunit component of native Kv4 channel complexes, regulating A-type currents and neuronal excitability in response to calcium fluctuations

• It is predominantly expressed at GABAergic synapses, suggesting important roles in inhibitory neurotransmission

• It functions as a Ca²⁺-dependent transcriptional repressor, binding to DRE (Downstream Regulatory Element) sites and controlling gene expression

• In Xenopus laevis embryos, it regulates the size of the neural plate by controlling neural progenitor proliferation

• It may play roles in GABAergic transmission and cell death mechanisms

The protein's diverse functions make it relevant to multiple research areas including neuroscience, developmental biology, and cancer research.

What are the available types of KCNIP1 antibodies and their sources?

Several manufacturers provide KCNIP1 antibodies with varying specifications:

• Sigma-Aldrich HPA022864: A rabbit-derived polyclonal antibody specifically designed for human KCNIP1 detection . This Prestige Antibody is extensively validated and characterized through the Human Protein Atlas project .

• Gentaur G-AB-06051: A rabbit polyclonal antibody with reactivity against human, mouse, and rat KCNIP1 . It was generated using a synthetic peptide of human KCNIP1 .

• Alomone Labs APC-141: A rabbit antibody that can be paired with their KChIP1/KCNIP1 Blocking Peptide (BLP-PC141) for specificity controls .

• Antibodies-online ABIN202072: A rabbit polyclonal antibody against the N-terminus of KCNIP1 with broad species reactivity including human, mouse, rat, cow, guinea pig, horse, chicken, monkey, pig, and Xenopus laevis .

When selecting an antibody, researchers should consider the required application, species reactivity, and availability of validation data for their specific experimental context.

What applications are KCNIP1 antibodies validated for?

KCNIP1 antibodies have been validated for several experimental applications:

• Western Blotting (WB): For detecting KCNIP1 protein in tissue or cell lysates

  • Sigma-Aldrich HPA022864: Recommended dilution 0.04-0.4 μg/mL

  • Alomone Labs APC-141: Validated for rat brain lysate analysis

• Immunohistochemistry (IHC): For visualizing KCNIP1 expression in tissue sections

  • Sigma-Aldrich HPA022864: Recommended dilution 1:20-1:50

  • Gentaur G-AB-06051: Recommended dilution 1:30-1:150

  • Antibodies-online ABIN202072: Validated for IHC including paraffin-embedded sections

• ELISA (Enzyme-Linked Immunosorbent Assay):

  • Gentaur G-AB-06051: Recommended dilution 1:5000-1:10000

The Human Protein Atlas project has extensively characterized the Sigma-Aldrich HPA022864 antibody through immunohistochemistry against hundreds of normal and disease tissues, and through immunofluorescence to map subcellular localization . This comprehensive validation provides confidence in antibody performance for tissue-level and subcellular studies.

How can I confirm the specificity of a KCNIP1 antibody?

Confirming antibody specificity is crucial for reliable experimental results. For KCNIP1 antibodies, several approaches should be considered:

• Blocking peptide controls: Use the immunizing peptide to pre-adsorb the antibody before application. For example, Alomone Labs provides the KChIP1/KCNIP1 Blocking Peptide (BLP-PC141) specifically for this purpose . When the Anti-KChIP1 antibody is preincubated with this peptide (recommended ratio: 1 μg peptide per 1 μg antibody), the signal should disappear in Western blot or immunohistochemistry applications if the antibody is specific .

• Knockout/knockdown controls: Tissues or cells lacking KCNIP1 expression provide excellent negative controls. The search results mention studies in Kcnip3 knockout mice that could provide a model for similar approaches with KCNIP1 .

• Cross-reactivity assessment: The KCNIP family has four members (KCNIP1-4) with significant sequence homology, particularly in their core domains . Antibodies targeting the more variable N-terminal regions are more likely to provide specific detection of KCNIP1 without cross-reactivity.

• Multiple antibodies approach: Using antibodies targeting different epitopes of KCNIP1 and observing concordant results increases confidence in specificity.

• Sequence alignment analysis: For example, the antibodies-online ABIN202072 antibody reports sequence identity analysis showing 100% identity across multiple species but only 92% for rabbit, suggesting potentially weaker reactivity with rabbit samples .

How can KCNIP1 antibodies be used to study the Kv4 channel complex?

KCNIP1 serves as a modulatory subunit of Kv4 potassium channels, making KCNIP1 antibodies valuable tools for studying these channel complexes:

• Structural interaction mapping: Crystal structure analyses have revealed that KCNIP1 interacts with Kv4 channels through multiple regions . The KChIP core domain possesses a hydrophobic crevice that may represent a binding pocket for hydrophobic residues in the proximal Kv4 N-terminus . Antibodies targeting specific epitopes can help map these interaction domains.

• Functional domain analysis: Studies have identified critical sites in three regions of Kv4.2 channels that are important for functional Kv4.2/KChIP interaction: the proximal N-terminus, the T1 domain, and the cytoplasmic C-terminus . KCNIP1 antibodies can be used in co-immunoprecipitation experiments to study how mutations in these regions affect Kv4/KCNIP1 interactions.

• Trafficking and localization studies: KCNIPs affect the surface expression and localization of Kv4 channels. Antibodies can be used in immunofluorescence or surface biotinylation assays to quantify changes in channel localization under various conditions.

• Immunoprecipitation studies: KCNIP1 antibodies can pull down the entire Kv4 channel complex, allowing identification of additional interacting partners or post-translational modifications.

Interestingly, studies have found some inconsistencies between binding and functional modulation. Certain T1-domain mutations in Kv4.2 still allowed KChIP1-induced modulation of inactivation while interfering with KChIP1 binding in yeast two-hybrid assays . This suggests that KChIP binding and coassembly may not be an absolute requirement for the observed KChIP-induced modulation of Kv4 channel gating in coexpression experiments .

What is known about KCNIP1's role in transcriptional regulation?

Recent research has revealed that KCNIP1 functions as a Ca²⁺-dependent transcriptional repressor:

• DNA binding capacity: KCNIP1 binds to DRE (Downstream Regulatory Element) sites in a Ca²⁺-dependent manner . This ability allows it to directly regulate gene expression.

• Developmental role: In Xenopus laevis embryos, KCNIP1 controls the size of the neural plate by regulating the proliferation of neural progenitors . It is expressed in the presumptive neural territories during embryonic development.

• Ca²⁺-dependency: The transcriptional repressor function of KCNIP1 is calcium-dependent, linking calcium signaling to gene expression regulation .

• Compensatory mechanisms: In vivo studies suggest the existence of compensatory mechanisms among KCNIP family members. While in cortico-hippocampal neurons from Kcnip3 knockout mice the expression levels of target genes are not significantly modified, additional invalidation of Kcnip2 results in significant increases in the expression of these genes . This suggests functional redundancy among KCNIP proteins as transcriptional regulators.

For investigating KCNIP1's transcriptional role, antibodies can be used in chromatin immunoprecipitation (ChIP) experiments to identify genomic binding sites, in immunofluorescence to track nuclear translocation, and in co-immunoprecipitation to identify interactions with other transcriptional regulators.

What is the significance of KCNIP1 expression in glioblastoma multiforme?

Analysis of KCNIP family gene expression in glioblastoma multiforme (GBM) reveals a distinctive pattern compared to normal brain tissues:

This differential expression pattern suggests specific roles for KCNIP1 in GBM pathophysiology . Several observations highlight the potential significance of this finding:

KCNIP1 antibodies could be valuable tools for investigating these aspects in GBM research, particularly for characterizing expression patterns in different tumor regions and in patient-derived samples.

What are the optimal conditions for using KCNIP1 antibodies in immunohistochemistry?

Successful immunohistochemistry with KCNIP1 antibodies requires careful optimization of several parameters:

How can I optimize Western blot protocols for KCNIP1 detection?

Optimizing Western blot protocols for KCNIP1 detection requires attention to several key factors:

• Sample preparation: For membrane-associated proteins like KCNIP1, effective extraction requires appropriate lysis buffers. Consider:

  • RIPA buffer for general extraction

  • Non-ionic detergents (e.g., Triton X-100) for gentler extraction

  • Inclusion of protease inhibitors to prevent degradation

  • Phosphatase inhibitors if studying phosphorylation status

• Gel percentage and running conditions: KCNIP1 has a molecular weight of approximately 28 kDa. Use 12-15% polyacrylamide gels for optimal resolution in this range.

• Transfer conditions: For proteins in this molecular weight range, semi-dry transfer (15-20V for 30-45 minutes) or wet transfer (100V for 1 hour) are both suitable.

• Blocking optimization: 5% non-fat milk in TBST is standard, but for phospho-specific detection, BSA may be preferable.

• Antibody dilutions: The Sigma-Aldrich HPA022864 antibody is recommended at 0.04-0.4 μg/mL for immunoblotting . Titrate to determine optimal concentration for your specific samples.

• Incubation conditions: Primary antibody incubation overnight at 4°C typically provides the best signal-to-noise ratio.

• Detection method: Enhanced chemiluminescence (ECL) is suitable for most applications, but more sensitive detection methods may be needed for low-abundance samples.

• Specificity controls: The Alomone Labs blocking peptide (BLP-PC141) can be used as a competitive inhibitor of antibody binding to confirm signal specificity . Their Western blot analysis of rat brain lysate demonstrates signal abolishment when the antibody is preincubated with the blocking peptide .

• Stripping and reprobing: If examining multiple proteins, gentle stripping conditions are recommended to preserve the membrane integrity for subsequent probing.

What considerations are important when using KCNIP1 antibodies for co-immunoprecipitation?

Co-immunoprecipitation (Co-IP) with KCNIP1 antibodies requires careful attention to preserve protein-protein interactions while ensuring specificity:

• Lysis buffer composition: Use gentle, non-denaturing buffers to preserve protein-protein interactions. Consider:

  • NP-40 or Triton X-100 based buffers (0.5-1%)

  • Physiological salt concentration (120-150 mM NaCl)

  • Protease and phosphatase inhibitors

• Calcium considerations: Since KCNIP1 is a calcium-binding protein, calcium levels can affect its interactions. Consider parallel experiments with:

  • Calcium-containing buffers (1-2 mM CaCl₂)

  • Calcium-free buffers with EGTA/EDTA to chelate calcium

  This approach can help distinguish calcium-dependent from calcium-independent interactions.

• Antibody selection: Choose antibodies with demonstrated specificity for immunoprecipitation. Consider epitope location - antibodies targeting functional domains might disrupt certain protein-protein interactions.

• Controls:

  • Input control: 5-10% of starting lysate

  • Negative control: Isotype-matched IgG

  • Blocking peptide control: Pre-incubate antibody with blocking peptide (e.g., Alomone Labs BLP-PC141)

  • When studying Kv4 channel interactions, include controls with Kv4 channel mutants known to disrupt KCNIP1 binding

• Crosslinking considerations: For weak or transient interactions, consider mild crosslinking with formaldehyde or DSP (dithiobis(succinimidyl propionate)).

• Elution conditions: For mass spectrometry applications, consider acid elution or on-bead digestion rather than boiling in SDS sample buffer.

• Reciprocal Co-IP: Confirm interactions by performing the reverse experiment (immunoprecipitate with antibodies against the interacting partner and blot for KCNIP1).

• Structural considerations: The crystal structure analysis of a KChIP1-Kv4.2 fusion protein has identified specific interaction regions . When studying Kv4 interactions, consider these known structural details in experimental design and interpretation.

How can I differentiate between KCNIP1 and other KCNIP family members?

Distinguishing KCNIP1 from other family members (KCNIP2-4) is crucial for experimental specificity. Several approaches can help achieve this differentiation:

• Antibody selection: The KCNIP family members differ primarily in their N-terminal regions . Antibodies targeting the N-terminus, such as the antibodies-online ABIN202072 antibody which specifically targets the N-Term of KCNIP1, offer better specificity .

• Molecular weight resolution: While all KCNIP proteins have similar molecular weights, careful gel resolution can help distinguish them:

  • KCNIP1: ~28 kDa

  • KCNIP2: ~30-32 kDa (depending on isoform)

  • KCNIP3: ~28-31 kDa (depending on isoform)

  • KCNIP4: ~28-32 kDa (depending on isoform)

  Use 12-15% polyacrylamide gels with extended running times for optimal separation.

• Peptide competition: Use specific blocking peptides, such as the KChIP1/KCNIP1 Blocking Peptide (BLP-PC141), which corresponds to amino acid residues 10-21 of rat KChIP1 (sequence: (C)SLQTKQRRPSKD) . This region may contain specific sequences that differ from other KCNIP family members.

• RNA-based approaches: For analyzing expression at the mRNA level, design primers targeting unique regions of each KCNIP family member.

• Expression patterns: Consider the differential expression patterns of KCNIP family members in different tissues. For example, in glioblastoma multiforme, KCNIP1 is upregulated while KCNIP2 and KCNIP3 are strongly downregulated .

• Knockout/knockdown controls: When available, use tissue or cells lacking specific KCNIP family members as controls for antibody specificity.

• Sequence alignment analysis: Compare the immunogen sequence of your antibody with corresponding regions in other KCNIP family members to predict potential cross-reactivity.

What are common pitfalls in KCNIP1 antibody experiments and how can they be addressed?

Several common challenges arise when working with KCNIP1 antibodies, each requiring specific troubleshooting approaches:

• Cross-reactivity with other KCNIP family members:

  • Problem: KCNIP1-4 share significant sequence homology.

  • Solution: Use antibodies targeting unique regions (typically N-terminal), perform peptide competition controls, and validate with knockout/knockdown samples when available.

• Calcium-dependent epitope accessibility:

  • Problem: As a calcium-binding protein, KCNIP1's conformation and epitope availability may change with calcium concentration.

  • Solution: Standardize calcium levels in buffers and consider testing both calcium-containing and calcium-free conditions to determine optimal detection conditions.

• Variable expression levels:

  • Problem: KCNIP1 expression varies across tissues and cell types.

  • Solution: Adjust protein loading amounts, antibody concentration, and exposure times based on expected expression levels. The differential expression in normal brain versus glioblastoma provides one example of this variability .

• Interference from post-translational modifications:

  • Problem: Modifications may mask epitopes or alter protein migration.

  • Solution: Use multiple antibodies targeting different epitopes and consider phosphatase treatment of samples if phosphorylation is suspected to affect detection.

• Non-specific bands in Western blots:

  • Problem: Additional bands may represent cross-reactivity, degradation products, or splice variants.

  • Solution: Include blocking peptide controls, optimize blocking conditions, and compare migration patterns with recombinant protein standards.

• Inconsistent results between binding and functional studies:

  • Problem: The search results mention inconsistencies between KChIP1 binding to Kv4.2 and functional effects on channel gating .

  • Solution: Use multiple, complementary techniques to address the same question and consider that protein interactions may not always correlate directly with functional effects.

• Subcellular localization challenges:

  • Problem: KCNIP1 may localize to different cellular compartments based on its dual roles in channel modulation and transcriptional regulation.

  • Solution: Use subcellular fractionation techniques and co-localization with compartment-specific markers to accurately determine localization patterns.

How can I optimize experiments to study KCNIP1's role in transcriptional regulation?

Investigating KCNIP1's function as a Ca²⁺-dependent transcriptional repressor requires specialized experimental approaches:

• Chromatin Immunoprecipitation (ChIP):

  • Use KCNIP1 antibodies to immunoprecipitate protein-DNA complexes

  • Include calcium dependency controls (with/without calcium or calcium chelators)

  • Target analysis of genes with DRE sites in their promoters

  • Consider ChIP-seq for genome-wide binding profile analysis

• Reporter assays:

  • Generate constructs with luciferase reporters driven by promoters containing DRE sites

  • Test transcriptional repression by KCNIP1 under varying calcium conditions

  • Include mutated DRE sites as specificity controls

• Nuclear translocation studies:

  • Use immunofluorescence with KCNIP1 antibodies to track potential calcium-dependent nuclear translocation

  • Combine with calcium imaging to correlate calcium fluctuations with localization changes

  • Include subcellular fractionation and Western blotting to quantify nuclear versus cytoplasmic distribution

• Gene expression analysis:

  • Compare expression of potential target genes in control versus KCNIP1-overexpressing or KCNIP1-knockdown systems

  • The study in Xenopus laevis embryos provides a model for examining KCNIP1's effect on neural progenitor proliferation

  • In GBM research, examine the relationship between KCNIP1 expression and the expression of prognosis genes with DRE sites in their promoters

• Protein-protein interactions:

  • Identify interactions with transcriptional machinery components using co-immunoprecipitation

  • Examine potential interactions with other transcriptional regulators

  • Consider calcium dependency of these interactions

• Calcium manipulation experiments:

  • Use calcium ionophores, chelators, or optogenetic tools to manipulate calcium levels

  • Correlate calcium changes with KCNIP1 transcriptional activity

  • The research showing KCNIP1 as a Ca²⁺-dependent transcriptional repressor provides a foundation for these approaches

• Disease-relevant models:

  • Given KCNIP1's upregulation in glioblastoma, patient-derived GBM cells or GBM cell lines provide relevant models for studying its transcriptional function in disease contexts

  • Compare KCNIP1 binding profiles and transcriptional effects between normal neural cells and GBM cells

How should changes in KCNIP1 expression be interpreted in disease contexts?

The altered expression of KCNIP1 in disease states, particularly its upregulation in glioblastoma multiforme, requires careful interpretation:

• Consider expression patterns of all KCNIP family members:

  • In GBM, KCNIP1 is upregulated while KCNIP2 and KCNIP3 are strongly downregulated

  • This pattern suggests a potential shift in the balance between family members rather than simply increased KCNIP function

  • The search results indicate possible compensatory mechanisms among KCNIP proteins

• Distinguish correlation from causation:

  • Altered expression may be a cause of pathology, a consequence, or an adaptive response

  • Functional studies manipulating KCNIP1 levels are needed to determine its causal role

  • The data suggesting a role of KCNIP proteins in stemness maintenance and dormant status of GBM stem-like cells points toward potential functional significance

• Consider dual functions:

  • Changes in KCNIP1 expression could affect ion channel modulation, transcriptional regulation, or both

  • Determine which function is most relevant to the disease context

  • In GBM, the presence of DRE sites in prognosis genes suggests transcriptional regulation may be particularly relevant

• Examine relationship to calcium signaling:

  • As a calcium-binding protein, KCNIP1 expression changes should be interpreted in the context of altered calcium homeostasis

  • The search results suggest that remodeling of Ca²⁺ homeostasis in GBM stem cells may contribute to altered expression of prognosis genes, potentially through KCNIP1

• Consider prognostic value:

  • While the search results indicate that high KCNIP2 expression correlates with reduced survival in GBM patients, similar data for KCNIP1 was not reported

  • Further studies correlating KCNIP1 expression with clinical outcomes could reveal potential prognostic value

• Therapeutic implications:

  • If KCNIP1 contributes to disease pathogenesis, it could represent a therapeutic target

  • Its calcium dependency provides a potential mechanism for modulating its activity

How can I interpret contradictory results between binding and functional studies of KCNIP1?

The search results highlight inconsistencies between binding studies and functional outcomes in KCNIP1-Kv4 channel research . These contradictions require careful interpretation:

• Recognize methodological limitations:

  • Binding assays (e.g., yeast two-hybrid) detect direct physical interactions but may miss functional effects

  • Electrophysiological studies measure functional outcomes but don't prove direct mechanisms

  • The search results note that "certain T1-domain mutations still allowed KChIP1-induced modulation of inactivation while interfering with KChIP1 binding"

• Consider multiple interaction sites:

  • The search results identify three regions of Kv4.2 important for KChIP interaction: the proximal N-terminus, the T1 domain, and the cytoplasmic C-terminus

  • Disruption of one interaction site may not eliminate all functional effects if multiple interaction sites exist

  • Crystal structure analyses have identified a hydrophobic crevice in the KChIP core domain that represents a binding pocket for hydrophobic residues in the proximal Kv4 N-terminus

• Evaluate indirect effects:

  • KCNIP1 might affect channel function through interactions with other proteins in the channel complex

  • The search results suggest that "KChIP binding and coassembly with Kv4 α-subunits may not be an absolute requirement for the observed KChIP-induced modulation"

• Consider native versus heterologous expression systems:

  • Studies in different expression systems may yield different results due to the presence or absence of additional regulatory factors

  • The search results mention using "naturally assembled and fully functional Kv4.2 channel constructs to study structural determinants for KChIP interaction"

• Integrate structural and functional data:

  • The X-ray structural analysis of a KChIP1-Kv4.2N30 fusion protein provides valuable insights into the physical interaction

  • Combine this structural information with functional studies to develop a more complete understanding of the relationship between binding and function

• Design experiments to directly address contradictions:

  • Use complementary techniques to study the same interaction

  • Include appropriate controls to rule out artifacts

  • Consider dose-dependency and kinetic aspects that might resolve apparent contradictions

What considerations are important when interpreting KCNIP1 localization data?

KCNIP1's dual roles in ion channel modulation and transcriptional regulation make interpretation of localization data particularly challenging:

• Expected subcellular distribution:

  • As a Kv channel interacting protein, KCNIP1 would be expected at the plasma membrane and in trafficking vesicles

  • As a transcriptional regulator, nuclear localization would be expected

  • The balance between these localizations may depend on cell type, developmental stage, and signaling state

• Calcium dependency:

  • KCNIP1's localization may change in response to calcium fluctuations

  • Its function as a Ca²⁺-dependent transcriptional repressor suggests calcium-dependent nuclear translocation

  • Consider calcium levels in fixation and staining protocols when interpreting immunohistochemistry results

• Antibody epitope considerations:

  • Antibodies targeting different epitopes may reveal different localization patterns if certain epitopes are masked in specific cellular compartments

  • Compare results from multiple antibodies targeting different regions of KCNIP1

• Distinguishing specific from non-specific staining:

  • Use blocking peptide controls such as the KChIP1/KCNIP1 Blocking Peptide (BLP-PC141)

  • Include genetic knockout/knockdown controls when available

  • Compare staining patterns across multiple antibodies

• Tissue-specific expression patterns:

  • The Human Protein Atlas project has mapped KCNIP1 expression across numerous normal and disease tissues

  • In normal brain versus GBM, KCNIP1 shows differential expression

  • Interpret localization data in the context of known tissue-specific expression patterns

• Developmental considerations:

  • The search results indicate that KCNIP1 is expressed in presumptive neural territories during Xenopus embryonic development

  • Localization patterns may change during development and differentiation

  • Consider the developmental stage when interpreting localization data

• Disease-related changes:

  • Altered localization in disease states may reflect pathological processes

  • In GBM, where KCNIP1 is upregulated, changes in subcellular localization might contribute to disease pathogenesis

  • Compare localization in normal versus disease tissues to identify disease-specific patterns