kctd15 Antibody

What is the molecular characterization of KCTD15 protein and how does it influence antibody selection?

KCTD15 (potassium channel tetramerisation domain containing 15) belongs to the KCTD family of proteins involved in diverse biological processes. The protein has significant roles in embryonic development through neural crest regulation and Wnt/β-catenin signaling pathway repression . KCTD15 has a calculated molecular weight of 32 kDa (283 amino acids), but typically appears at approximately 26 kDa in experimental conditions . This discrepancy is critical to consider when validating antibody specificity in Western blot applications.

When selecting an appropriate KCTD15 antibody, researchers should consider:

What experimental strategies can effectively validate KCTD15 antibody specificity?

Methodical validation of KCTD15 antibodies is essential for reliable experimental outcomes. Consider implementing these validation approaches:

• Western blot validation:

  • Compare observed band (~26 kDa) with expected molecular weight

  • Include positive control tissues/cells: mouse brain tissue, C6 cells, HEK-293 cells, or mouse lung tissue

  • Include KCTD15 knockdown/knockout samples as negative controls

• Immunohistochemistry optimization:

  • Test recommended antigen retrieval methods: TE buffer pH 9.0 or citrate buffer pH 6.0

  • Validate with known positive tissue controls (e.g., mouse spleen tissue)

  • Perform antibody titration within the recommended range (1:50-1:500)

• Expression manipulation controls:

  • Compare antibody detection between wild-type and KCTD15 knockdown samples

  • Use KCTD15 overexpression systems (e.g., transfected 293T cells) as positive controls

  • For inducible systems, include non-induced controls

Each validation method contributes to confirming antibody specificity and optimizing experimental conditions for your particular application and biological system.

What are the optimal conditions for KCTD15 detection across different experimental techniques?

The following application-specific parameters have been experimentally validated for KCTD15 detection:

Western Blot Optimization:

Immunohistochemistry Protocol:

Immunoprecipitation Guidelines:

These optimized conditions provide a starting point that should be further refined for your specific experimental system and research objectives.

How can researchers most effectively design KCTD15 knockdown and overexpression experiments?

Based on published KCTD15 functional studies, the following experimental design strategies are recommended:

• Expression manipulation approaches:

  • For transient manipulation: standard transfection with KCTD15 expression vectors (e.g., pCMV6-AC vector)

  • For stable manipulation: lentiviral or retroviral transduction

  • For inducible systems: Tet-inducible expression systems have been validated in vivo

• Verification methods:

  • Transcriptional level: qRT-PCR to confirm altered KCTD15 mRNA expression

  • Protein level: Western blot using validated antibodies (1:500-1:1000 dilution)

• Functional assessment metrics:

  • Proliferation: EdU incorporation, colony formation assays

  • Proliferation markers: Ki67 and PCNA protein levels by Western blot or IHC

  • Apoptosis: Annexin V-FITC/PI double staining by flow cytometry

  • Apoptosis markers: cleaved caspase 3, cleaved caspase 9, and p53 by Western blot

• In vivo model considerations:

  • Use inducible expression systems (e.g., Tet-inducible) for temporal control

  • Include appropriate vehicle controls

  • Monitor tumor growth measurements and analyze Ki67 expression by IHC in tumor tissues

These methodological approaches provide a comprehensive framework for investigating KCTD15 function in various biological contexts.

How does KCTD15 function as an anti-tumor factor in colorectal cancer, and what experimental evidence supports this role?

Recent research has established KCTD15 as a significant anti-tumor factor in colorectal cancer (CRC) through multiple lines of experimental evidence:

• Expression patterns in clinical samples:

  • KCTD15 is significantly downregulated in CRC tissues compared to normal tissues across multiple datasets (GSE146587, GEPIA-COAD, GEPIA-READ)

  • This consistent downregulation pattern suggests a potential tumor suppressor role

• In vitro functional evidence:

  • KCTD15 overexpression in CRC cells resulted in:

    • Decreased cell viability measured by MTT assay

    • Reduced EdU-positive cells indicating decreased DNA synthesis

    • Inhibited colony formation capacity

    • Downregulation of proliferation markers Ki67 and PCNA

    • Increased apoptosis measured by Annexin V-FITC/PI staining

    • Elevated apoptosis markers (cleaved caspase 3, cleaved caspase 9, and p53)

• In vivo validation:

  • Induction of KCTD15 expression significantly inhibited tumor growth in mouse models

  • Reduced Ki67 expression was observed in tumor tissues, confirming the anti-proliferative effect

• Molecular regulation:

  • KCTD15 expression is orchestrated by the FTO-YTHDF2 axis, suggesting complex regulatory mechanisms

These findings collectively establish KCTD15 as a tumor suppressor in CRC through dual mechanisms of proliferation inhibition and apoptosis promotion.

What is the relationship between KCTD15 and the NF-κB signaling pathway, and how can researchers investigate this interaction?

Research has uncovered a significant relationship between KCTD15 and the NF-κB signaling pathway, particularly in hematological malignancies:

• Pathway interaction evidence:

  • KCTD15 upregulation is associated with increased NF-κB signaling activity

  • Silencing of KCTD15 in MLL-rearranged leukemia models attenuated the NF-κB pathway

  • This attenuation was associated with downregulation of phosphorylated IKK-β and phosphorylated IκB-α

• Molecular mechanism:

  • KCTD15 physically interacts with IKK-β, as demonstrated by:

    • Proximity ligation assays

    • Immunoprecipitation experiments

  • KCTD15 upregulates IKK-β, which provides a mechanistic basis for its effect on NF-κB signaling

• Functional validation approaches:

  • Luciferase reporter assays confirmed the association between KCTD15 expression and NF-κB activation

• Experimental investigation strategies:

  • Co-immunoprecipitation to confirm physical interaction between KCTD15 and pathway components

  • Phosphorylation analysis of IKK-β and IκB-α following KCTD15 manipulation

  • NF-κB nuclear translocation assessment using nuclear/cytoplasmic fractionation or immunofluorescence

  • NF-κB target gene expression analysis after KCTD15 modulation

This KCTD15-NF-κB relationship may have broader implications for understanding KCTD15's role in various physiological and pathological conditions beyond leukemia, including neuronal development, cancer progression, and metabolic disorders .

How can researchers effectively use KCTD15 antibodies to investigate its role in developmental processes?

KCTD15 has documented roles in embryonic development, particularly in neural crest regulation through Wnt/β-catenin pathway repression . Researchers can employ the following methodological approaches to investigate developmental roles:

• Developmental expression analysis:

  • Temporal expression profiling using Western blot with standardized tissue amounts across developmental stages

  • Spatial expression mapping using immunohistochemistry with optimized antigen retrieval (TE buffer pH 9.0)

  • Co-localization studies with developmental markers using dual immunofluorescence

• Pathway interaction investigation:

  • Co-immunoprecipitation of KCTD15 with Wnt/β-catenin pathway components

  • Proximity ligation assays to detect protein-protein interactions in situ

  • Combined KCTD15 immunostaining with pathway activity reporters

• Functional developmental studies:

  • Temporal manipulation of KCTD15 expression using inducible systems

  • Tissue-specific KCTD15 modulation using Cre-Lox approaches

  • Assessment of developmental phenotypes following KCTD15 manipulation

• Antibody selection considerations:

  • Species compatibility with model organism (human, mouse, rat reactivity available)

  • Application-specific optimization (WB: 1:500-1:1000; IHC: 1:50-1:500)

  • Validation in developmental tissues using knockout/knockdown controls

These methodological approaches provide a framework for investigating KCTD15's developmental roles while leveraging the specificity and sensitivity of well-validated KCTD15 antibodies.

What are common technical challenges when working with KCTD15 antibodies and how can researchers address them?

Researchers may encounter several technical challenges when working with KCTD15 antibodies. The following troubleshooting strategies address common issues:

• Western blot detection issues:

  • Problem: Multiple bands or incorrect molecular weight
    Solution: Verify expected molecular weight (26 kDa observed vs. 32 kDa calculated) ; optimize antibody dilution (1:500-1:1000); include positive control tissues (mouse brain/lung)

  • Problem: Weak or no signal
    Solution: Increase protein loading (35 μg successful for HeLa cells) ; optimize transfer conditions; ensure appropriate blocking buffer; verify sample expresses KCTD15 (C6, HEK-293 cells as positive controls)

• Immunohistochemistry optimization:

  • Problem: High background staining
    Solution: Optimize antibody dilution (start with 1:50-1:500 range) ; modify blocking conditions; extend washing steps

  • Problem: Weak or no signal
    Solution: Test both recommended antigen retrieval methods (TE buffer pH 9.0 or citrate buffer pH 6.0) ; optimize incubation time and temperature; verify with positive control tissue (mouse spleen)

• Cross-reactivity considerations:

  • Problem: Potential cross-reactivity with other KCTD family proteins
    Solution: Validate with KCTD15 knockdown/knockout controls; compare staining patterns with different KCTD15 antibodies targeting distinct epitopes

• Species compatibility:

  • Problem: Uncertain cross-reactivity with specific model organism
    Solution: Verify tested reactivity (human, mouse, rat) ; conduct preliminary validation in your specific species; consider epitope conservation analysis

These systematic troubleshooting approaches can help researchers optimize KCTD15 antibody performance across various applications and experimental systems.

How can researchers integrate KCTD15 antibody-based techniques with other methodologies to comprehensively study its biological functions?

A multi-methodological approach yields the most comprehensive understanding of KCTD15 biology. Consider these integrated research strategies:

• Combining protein-level and transcriptional analyses:

  • Correlate KCTD15 protein levels (Western blot, IHC) with mRNA expression (qRT-PCR, RNA-seq)

  • Investigate post-transcriptional regulation mechanisms using antibody-based detection of protein levels

  • Example methodology: Western blot (1:500-1:1000 dilution) paired with qRT-PCR as demonstrated in colorectal cancer studies

• Integrating functional and mechanistic studies:

  • Connect KCTD15 expression levels with functional outcomes and pathway activity

  • Methodological approach: combine KCTD15 immunodetection with:

    • Proliferation assays (EdU incorporation, Ki67 staining)

    • Apoptosis assessment (Annexin V-FITC/PI staining)

    • NF-κB pathway activity (phospho-IKK-β and phospho-IκB-α levels)

• Protein interaction networks:

  • Identify KCTD15 binding partners and functional complexes

  • Integrated techniques:

    • Immunoprecipitation using optimized conditions (0.5-4.0 μg antibody per 1.0-3.0 mg lysate)

    • Mass spectrometry identification of co-precipitated proteins

    • Proximity ligation assays for in situ interaction verification

    • Co-localization studies via multi-channel immunofluorescence

• In vivo and in vitro correlation:

  • Connect tissue-level and cellular findings

  • Approach: pair in vivo IHC detection of KCTD15 in tumor models with in vitro mechanistic studies using the same antibody

This integrated approach provides a comprehensive framework for elucidating KCTD15's biological roles across multiple scales of biological organization and disease contexts.

What novel KCTD15 functions are being discovered, and how can researchers effectively investigate these emerging roles?

Recent research has begun uncovering novel KCTD15 functions beyond its established roles, opening several promising research directions:

• KCTD15 in metabolic regulation:

  • References suggest potential implications in obesity/diabetes

  • Investigation approach:

    • Analyze KCTD15 expression in metabolic tissues using optimized IHC protocols (1:50-1:500 dilution)

    • Study metabolic phenotypes in KCTD15 knockout/transgenic models

    • Explore interactions with metabolic signaling pathways

• KCTD15 in neuronal development:

  • KCTD15's role in neural crest development suggests broader neuronal functions

  • Research strategy:

    • Characterize KCTD15 expression in developing and mature neural tissues

    • Investigate neurodevelopmental effects of KCTD15 manipulation

    • Study potential interactions with neurodevelopmental signaling networks

• KCTD15 in cancer beyond colorectal and hematological malignancies:

  • Given its tumor suppressor role in colorectal cancer and involvement in leukemia , KCTD15 may have broader cancer relevance

  • Investigation methodology:

    • Pan-cancer expression analysis using validated antibodies

    • Correlation of KCTD15 levels with clinical outcomes

    • Functional studies in additional cancer types using established protocols

• KCTD15 in immune regulation:

  • The connection to NF-κB signaling suggests potential immunological functions

  • Experimental approach:

    • Analyze KCTD15 expression in immune cells and tissues (spleen tissue has shown positive IHC)

    • Study immune phenotypes in KCTD15-modified models

    • Investigate KCTD15-dependent immune signaling pathways

These emerging research directions can be effectively investigated using well-validated KCTD15 antibodies in combination with the methodological approaches detailed throughout this FAQ document.