KCTD15 PAT2B11AT Antibody

What is KCTD15 and what are its known biological functions?

KCTD15 (Potassium Channel Tetramerization Domain Containing 15) is a protein encoded by the KCTD15 gene in humans. Also known as BTB/POZ domain-containing protein KCTD15, it serves several important biological functions:

• During embryonic development, it is involved in neural crest formation

• It inhibits AP2 transcriptional activity by interacting with its activation domain

• It is highly expressed in the brain and hypothalamus

• It has been identified as a genetic locus linked to higher than normal BMI in humans, along with genes such as GNPDA2, MTCH2, FTO, and TMEM18

• Recent research has revealed its significant overexpression in certain breast cancer subtypes, particularly in HER2+ breast cancer patients

What epitope does the PAT2B11AT antibody recognize on KCTD15?

The PAT2B11AT antibody specifically recognizes an epitope within amino acids 1-159 of the human KCTD15 protein. This monoclonal antibody was generated by immunizing BALB/c mice with a recombinant human KCTD15 protein fragment (amino acids 1-159) purified from E. coli . The antibody was derived from the hybridization of mouse F0 myeloma cells with spleen cells from the immunized mice, resulting in a mouse IgG2b heavy chain and kappa light chain antibody that specifically targets the N-terminal region of KCTD15 .

What experimental applications has the KCTD15 PAT2B11AT antibody been validated for?

The KCTD15 PAT2B11AT antibody has been validated for multiple research applications, making it versatile for KCTD15 studies:

• ELISA (Enzyme-Linked Immunosorbent Assay)

• Western blot analysis

• Flow cytometry

• ICC/IF (Immunocytochemistry/Immunofluorescence)

Researchers should note that while the antibody has been tested for specificity and reactivity in these applications, optimal conditions may vary between laboratories. Each investigation should include appropriate titration of the reagent to obtain optimal results for the specific experimental system being used .

What are the optimal storage and handling conditions for this antibody?

To maintain optimal efficacy of the KCTD15 PAT2B11AT antibody, researchers should follow these storage and handling guidelines:

• For short-term storage (up to 1 month): Store at 4°C

• For long-term storage: Store at -20°C

• Avoid freeze-thaw cycles, which can degrade antibody quality and functionality

• The antibody has a shelf life of approximately 12 months at -20°C and 1 month at 4°C

• The antibody is formulated at 1mg/ml in PBS (pH 7.4) with 10% Glycerol and 0.02% Sodium Azide

Proper storage conditions are critical for maintaining antibody performance across multiple experiments and ensuring reproducible results.

How can I confirm the specificity of the KCTD15 PAT2B11AT antibody in my experimental system?

To validate the specificity of the KCTD15 PAT2B11AT antibody in your experimental system, consider implementing these methodological approaches:

• Positive control testing: Utilize cell lines known to express high levels of KCTD15, such as SKBR3 breast cancer cells

• Negative control testing: Include cells with low KCTD15 expression (e.g., normal breast cell lines like MCF10A) for comparison

• Western blot validation: Confirm detection of a single band at the expected molecular weight for KCTD15

• Knockdown validation: Test the antibody on wild-type vs. KCTD15-silenced cells using siRNA or CRISPR methods

• Blocking experiments: Pre-incubate the antibody with the immunizing peptide (amino acids 1-159) to confirm specific binding

• Cross-reactivity assessment: Test against closely related proteins to ensure specificity to KCTD15

What is the significance of KCTD15 overexpression in HER2+ breast cancer?

Recent research has revealed critical insights into KCTD15's role in breast cancer, particularly in HER2+ subtypes:

• Studies demonstrate significant KCTD15 overexpression in Luminal A, Luminal B, and especially HER2+ breast cancer patients compared to healthy controls

• The SKBR3 cell line (a model system for HER2+ breast cancer) shows remarkably higher KCTD15 expression compared to other breast cancer cell lines and normal breast epithelial cells (MCF10A)

• KCTD15 silencing in SKBR3 cells using CRISPR/Cas9 technology significantly attenuates cell proliferation and cell cycle progression

• KCTD15 silencing also sensitizes HER2+ breast cancer cells to the cytotoxic agent doxorubicin, suggesting a potential role in resistance mechanisms

These findings collectively indicate that KCTD15 may play an active role in HER2+ breast cancer carcinogenesis, suggesting its potential as both a biomarker and therapeutic target for this aggressive breast cancer subtype .

What methodological approaches are recommended for studying KCTD15's role in cell proliferation?

To investigate KCTD15's impact on cell proliferation, particularly in cancer models, researchers should consider these methodological approaches:

• CRISPR/Cas9 gene editing: This approach has been successfully used to silence KCTD15 in breast cancer cell lines, allowing for precise study of its impact on proliferation

• Cell proliferation assays: Methods such as MTT/XTT, BrdU incorporation, or real-time cell analysis can quantify changes in proliferation after KCTD15 manipulation

• Cell cycle analysis: Flow cytometry-based analysis of cell cycle distribution following KCTD15 silencing can reveal its impact on cell cycle progression

• Colony formation assays: These assess the long-term effects of KCTD15 modulation on cell growth and survival

• Protein interaction studies: Co-immunoprecipitation to identify KCTD15 binding partners involved in proliferation pathways

• Transcriptional profiling: RNA-seq analysis of cells with modulated KCTD15 expression to identify affected pathways

In experimental design, it's essential to include appropriate controls and consider cell line-specific contexts, as KCTD15 expression varies significantly across different breast cancer subtypes .

How can I optimize detection of KCTD15 across different breast cancer cell lines?

Optimizing KCTD15 detection across various breast cancer cell lines requires consideration of several methodological factors:

• Expression level variation: Be aware that KCTD15 expression varies significantly across breast cancer subtypes:

  • HER2+ cell lines (e.g., SKBR3): Highest expression

  • Luminal A and B cell lines: Moderate to high expression

  • Normal breast epithelial lines (e.g., MCF10A): Low expression

• Antibody titration: The recommended starting dilution for PAT2B11AT antibody is 1:250-500 for most applications , but optimal concentration should be determined for each cell line

• Detection method selection:

  • Western blotting: Optimize protein extraction using buffers containing appropriate detergents with protease inhibitors

  • Flow cytometry: Establish gating strategies based on negative controls

  • ICC/IF: Compare different fixation methods to determine optimal epitope preservation

• Signal amplification: Consider signal enhancement techniques for low-expressing cell lines

• Quantification approach: Use digital image analysis for immunofluorescence or densitometry for Western blots to enable objective comparison across cell lines

What are appropriate controls for KCTD15 expression studies in breast cancer research?

When conducting KCTD15 expression studies in breast cancer research, implement these essential controls:

Positive Controls:

• SKBR3 cells (HER2+ breast cancer cell line) which consistently show high KCTD15 expression

• Luminal A and B breast cancer cell lines that demonstrate significant KCTD15 expression

• Brain or hypothalamus tissue samples, where KCTD15 is naturally highly expressed

• HER2+ breast cancer tissue sections for immunohistochemistry studies

Negative Controls:

• MCF10A normal breast epithelial cells (showing very low KCTD15 expression)

• KCTD15 knockout/knockdown cell lines generated via CRISPR-Cas9 or siRNA technology

• Isotype control antibodies matched to the primary antibody class and species

• Secondary antibody-only controls to assess background staining

• Internal tissue controls with mild KCTD15 expression (normal ductal-lining epithelium) for IHC studies

Methodological Controls:

• Include gradient expression controls when possible (cell lines with known low, medium, and high expression)

• For quantitative analyses, include calibration standards of known KCTD15 concentrations

• Technical replicates to assess method reproducibility

• Biological replicates to account for natural variation

What techniques are most effective for KCTD15 gene silencing in breast cancer research?

Based on published research, several techniques have proven effective for KCTD15 gene silencing in breast cancer research:

CRISPR/Cas9 Gene Editing:

• Most effective for complete and stable KCTD15 knockout

• Successfully employed in SKBR3 cells (HER2+ breast cancer model)

• Enables generation of stable cell lines for long-term studies

• Provides precise genomic modifications with minimal off-target effects when designed properly

RNA Interference (RNAi):

• siRNA: Effective for transient KCTD15 knockdown (3-7 days)

• shRNA: Suitable for longer-term knockdown when delivered via lentiviral vectors

• Provides flexibility in targeting different regions of KCTD15 mRNA

Methodological Considerations:

• Include scrambled or non-targeting controls with similar GC content

• Validate knockdown efficiency using both qRT-PCR (mRNA level) and Western blot (protein level)

• For breast cancer studies, optimize transfection/transduction protocols specific to the cell line being used

• Consider rescue experiments with KCTD15 overexpression to confirm specificity of observed effects

The choice of silencing technique should be based on experimental duration, desired knockdown efficiency, and specific research questions.

How does KCTD15 interact with the AP2 transcription factor pathway?

KCTD15 has been identified as an inhibitor of AP2 transcriptional activity through interaction with its activation domain . To study this interaction and its functional consequences, researchers can employ these methodological approaches:

Molecular Interaction Studies:

• Co-immunoprecipitation (Co-IP): Use anti-KCTD15 antibodies to pull down protein complexes and probe for AP2

• Proximity ligation assay (PLA): Visualize and quantify KCTD15-AP2 interactions in situ

• Yeast two-hybrid or mammalian two-hybrid assays: Map specific interaction domains

• GST pull-down assays with recombinant proteins: Determine if interaction is direct or requires cofactors

Functional Analysis:

• Luciferase reporter assays: Measure AP2 transcriptional activity in the presence/absence of KCTD15

• ChIP-seq: Analyze AP2 binding to target genes with and without KCTD15 expression

• RNA-seq: Identify genes differentially regulated by AP2 when KCTD15 is modulated

• KCTD15 domain mutation analysis: Identify which domains are critical for AP2 inhibition

Physiological Relevance:

• Correlate KCTD15-AP2 interaction with cancer phenotypes (proliferation, migration, etc.)

• Investigate how this interaction may contribute to breast cancer development and progression

This interaction may be particularly relevant in HER2+ breast cancer, where KCTD15 is highly expressed , and understanding it could reveal potential therapeutic targets in the AP2 signaling pathway.

What is the relationship between KCTD15 expression and chemotherapy sensitivity in breast cancer?

Recent research has revealed an important relationship between KCTD15 expression and chemotherapy sensitivity in breast cancer:

• KCTD15 silencing in SKBR3 cells (HER2+ breast cancer model) sensitizes them to the cytotoxic agent doxorubicin, suggesting KCTD15 may contribute to chemoresistance mechanisms

• This relationship appears particularly significant in HER2+ breast cancers, where KCTD15 is highly overexpressed

Methodological Approaches to Study This Relationship:

• Drug sensitivity assays: Compare IC50 values of chemotherapeutic agents in KCTD15-expressing vs. KCTD15-silenced cells

• Apoptosis assays: Measure changes in apoptotic response to chemotherapy following KCTD15 manipulation

• Mechanistic investigations: Explore whether KCTD15 affects drug efflux pumps, DNA damage repair pathways, or anti-apoptotic mechanisms

• Patient-derived xenografts: Assess correlation between KCTD15 expression and treatment response in clinically relevant models

• Clinical sample analysis: Retrospectively analyze KCTD15 expression in relation to treatment outcomes in breast cancer patients

Understanding this relationship could lead to the development of KCTD15 as a predictive biomarker for chemotherapy response or as a target for combination therapies aimed at overcoming resistance in HER2+ breast cancer .

What quantitative methods are recommended for assessing KCTD15 expression in clinical samples?

Several quantitative methods can be employed to assess KCTD15 expression in clinical samples, each with specific advantages for research applications:

Immunohistochemistry (IHC):

• Allows visualization of KCTD15 expression in tissue context

• Can be performed on formalin-fixed paraffin-embedded (FFPE) tissues from archives

• Recommended scoring system: Assess staining intensity (0=negative, 1=weak, 2=moderate, 3=strong) and percentage of positive cells

• Compare with normal ductal-lining epithelium as an internal control

• Advantages: Preserves tissue architecture, enables retrospective studies

• Limitations: Semi-quantitative, subject to interpreter variability

Quantitative Real-Time PCR (qRT-PCR):

• Measures KCTD15 mRNA expression levels

• Use validated reference genes appropriate for breast tissue

• Calculate relative expression using the 2^-ΔΔCt method

• Advantages: Highly sensitive, specific, good dynamic range

• Limitations: Requires fresh or properly preserved tissue, does not assess protein expression

Western Blot:

• Quantifies KCTD15 protein expression

• Use PAT2B11AT antibody at 1:250-500 dilution

• Normalize to appropriate loading controls

• Advantages: Confirms protein size, semi-quantitative

• Limitations: Requires significant amount of fresh/frozen tissue

Flow Cytometry:

• Allows single-cell quantification of KCTD15 expression

• Recommended for analysis of disaggregated tumor samples

• Advantages: Provides detailed expression distribution, can be combined with other markers

• Limitations: Requires viable cells, labor-intensive sample preparation

For clinical applications, IHC is most commonly used due to practicality with FFPE samples, while research applications may benefit from combining multiple approaches for comprehensive analysis.

What technical challenges should researchers anticipate when using this antibody for immunohistochemistry?

Researchers may encounter several technical challenges when using KCTD15 antibodies for immunohistochemistry:

Epitope Accessibility:

• Formalin fixation can mask the KCTD15 epitope

• Solution: Optimize antigen retrieval methods (heat-induced epitope retrieval with appropriate buffers)

• Test different retrieval times and temperatures to determine optimal conditions

Expression Heterogeneity:

• KCTD15 expression varies significantly across breast cancer subtypes

• Solution: Include known positive controls (HER2+ tissues) and negative controls with each staining batch

• Use hyperplastic areas with mild positivity in normal ductal-lining epithelium as internal controls

Background Staining:

• Non-specific binding can complicate interpretation

• Solution: Optimize blocking procedures (5% normal serum, 1% BSA)

• Titrate primary antibody concentration (starting at 1:250 dilution)

• Consider mouse-on-mouse blocking if using mouse antibodies on mouse tissues

Signal Optimization:

• Weak signal in low-expressing samples

• Solution: Consider signal amplification systems

• Optimize incubation times and temperatures

• Use fresher tissue sections when possible

Reproducibility:

• Batch-to-batch variability in staining intensity

• Solution: Include standard reference tissues in each run

• Standardize all protocol steps (fixation time, section thickness, staining conditions)

Quantification Standardization:

• Consistent scoring across different samples and observers

• Solution: Establish clear scoring criteria based on comparison with internal controls

• Consider digital image analysis for more objective quantification

How does KCTD15 expression vary across breast cancer molecular subtypes?

Research has revealed significant variations in KCTD15 expression across breast cancer molecular subtypes, with important biological and clinical implications:

Expression Pattern Across Subtypes:

• HER2+ subtype: Shows the highest level of KCTD15 overexpression among breast cancer subtypes

• Luminal A and Luminal B subtypes: Demonstrate significant but generally lower KCTD15 overexpression compared to HER2+

• Normal breast tissue: Exhibits very mild positivity in normal ductal-lining epithelium

Research and Clinical Implications:

• Diagnostic potential:

  • KCTD15 could serve as an additional marker in breast cancer subtyping

  • May help identify specific HER2+ tumors with more aggressive phenotypes

• Therapeutic relevance:

  • HER2+ tumors with high KCTD15 expression may benefit from targeted approaches

  • KCTD15 silencing sensitizes HER2+ cells to doxorubicin, suggesting potential for combination therapies

  • Could inform treatment selection in precision oncology approaches

• Biological insights:

  • The pattern of expression suggests KCTD15 may interact with HER2 signaling pathways

  • Understanding the mechanisms behind subtype-specific overexpression could reveal new oncogenic pathways

  • KCTD15 silencing significantly attenuates proliferation and cell cycle progression in HER2+ cells

• Methodological considerations:

  • When studying KCTD15, researchers should select appropriate cell line models based on the subtype of interest

  • Subtype-specific expression patterns should be considered when designing experiments and interpreting results

  • Validation across multiple cell lines representing different subtypes is recommended