KCTD12 Antibody

What cell and tissue types express KCTD12 that can be detected with KCTD12 antibody?

KCTD12 protein expression has been successfully detected in multiple tissue and cell types using appropriate antibodies. Positive Western blot detection has been confirmed in mouse, rat, and human brain tissues, as well as in several commonly used cell lines including MCF-7, HEK-293, and HeLa cells . For immunohistochemistry applications, KCTD12 has been successfully detected in human Bowen disease specimens, intrahepatic cholangiocarcinoma tissue, and stomach tissue . These findings indicate that KCTD12 is expressed across diverse tissue types, making the antibody useful for comparative studies across different experimental models.

What are the recommended dilutions for KCTD12 antibody across different experimental applications?

Optimal dilution of KCTD12 antibody varies significantly depending on the application technique. For Western blot analyses, a dilution range of 1:500-1:2000 is recommended . Immunohistochemistry applications typically require a higher concentration with recommended dilutions between 1:50-1:500 . For immunofluorescence and immunocytochemistry applications, an even higher concentration may be needed with suggested dilutions of 1:10-1:100 . It is strongly advised that researchers perform antibody titration experiments within their specific experimental systems to determine the optimal concentration that provides maximum signal with minimal background.

What are the critical controls needed when using KCTD12 antibody for the first time?

When validating KCTD12 antibody for your research, several controls are essential:

• Positive control: Include samples known to express KCTD12, such as brain tissue or HeLa cells .

• Negative control: Use tissue/cells where KCTD12 is not expressed or include KCTD12 knockdown samples.

• Peptide competition assay: Pre-incubation of the antibody with excess KCTD12 peptide should abolish specific staining.

• Secondary antibody control: Omit primary antibody to assess non-specific binding of the secondary antibody.

• Cross-reactivity assessment: Test the antibody in samples from multiple species if cross-species detection is claimed.

For knockdown validation, techniques similar to those used in KCTD12 functional studies, such as siRNA approaches demonstrated in HT29 and GIST T1 cell lines, can be employed .

How can KCTD12 antibody be effectively used to study the relationship between KCTD12 and cancer stem cell markers?

To investigate KCTD12's relationship with cancer stem cell markers, researchers should use a multi-technique approach:

• Co-immunostaining method: Perform double immunofluorescence staining with KCTD12 antibody (1:10-1:100 dilution) alongside antibodies against established cancer stem cell markers such as CD44, CD133, and CD29 . This allows visualization of potential co-localization or exclusive expression patterns.

• Flow cytometry correlation: After KCTD12 knockdown or overexpression, quantitative assessment of stem cell marker expression can be performed. As demonstrated in previous studies, KCTD12 silencing in HT29 cells significantly increased the percentage of CD44+ and CD133+ cells, which can be measured by flow cytometry .

• Western blot validation: Following experimental manipulation of KCTD12 levels, changes in stem cell marker expression should be confirmed via Western blot. Previous research showed that silencing and overexpression of KCTD12 were capable of increasing and decreasing, respectively, the expression of CD44, CD133, and CD29 at both protein and mRNA levels .

• Functional assays: Complement antibody-based detection with functional assays like sphere formation assays, which demonstrated that KCTD12 knockdown increased sphere size in HT29 cells .

What are the troubleshooting approaches when KCTD12 antibody shows non-specific binding or background issues?

When encountering non-specific binding or high background with KCTD12 antibody, consider these methodological solutions:

• Optimization of antigen retrieval for IHC: For KCTD12 detection in tissues, TE buffer at pH 9.0 is recommended, although citrate buffer at pH 6.0 is an acceptable alternative . Suboptimal antigen retrieval can lead to false negative or high background.

• Blocking optimization: Increase blocking time and concentration or try alternative blocking reagents (BSA, normal serum, commercial blockers) to reduce non-specific binding.

• Antibody dilution adjustment: Test a broader range of dilutions than the recommended range. For problematic samples, start with higher dilutions (1:2000-1:5000 for WB) and titrate to find the optimal signal-to-noise ratio.

• Sequential double incubation: For difficult tissues, consider incubating with primary antibody, washing thoroughly, then repeating primary antibody incubation before proceeding to secondary antibody.

• Validate specificity with knockdown controls: Use KCTD12 siRNA-treated samples as negative controls, similar to the approaches used in functional studies of KCTD12 in colorectal cancer and GIST cells .

• Cross-adsorption of antibody: If cross-reactivity with related KCTD family proteins is suspected, pre-adsorb the antibody with recombinant proteins of related family members.

How can researchers effectively monitor KCTD12 expression changes during ERK pathway modulation experiments?

To accurately monitor KCTD12 expression changes during ERK pathway modulation:

• Time course Western blot analysis: Treat cells with ERK1/2 inhibitor U0126 at different time points (1h, 3h, 6h, 12h, 24h) and use KCTD12 antibody (1:500-1:2000 dilution) to detect expression changes via Western blot . Include phospho-ERK and total ERK antibodies in parallel blots to confirm pathway inhibition.

• Quantitative immunofluorescence: Use KCTD12 antibody (1:10-1:100) for immunofluorescence to visualize subcellular localization changes following ERK pathway modulation . Quantify signal intensity using appropriate imaging software.

• Co-immunoprecipitation studies: Employ KCTD12 antibody for immunoprecipitation followed by Western blot for ERK pathway components to detect potential protein-protein interactions that may explain the regulatory relationship.

• Validation in multiple cell models: Previous research has shown KCTD12 suppresses CRC cell stemness markers by inhibiting the ERK pathway, as ERK1/2 inhibitor U0126 abolished the increase in expression of CRC cell stemness markers induced by KCTD12 downregulation . This approach should be replicated across different cell types to establish broader biological relevance.

• Correlation with functional outcomes: Combine antibody detection with functional assays such as colony formation or spheroid formation to correlate KCTD12 expression levels with biological effects of ERK pathway modulation.

What experimental approaches can demonstrate the regulatory relationship between KIT and KCTD12 using antibody-based detection methods?

Based on findings that KIT negatively regulates KCTD12 in gastrointestinal stromal tumors , researchers can design experiments to investigate this relationship:

• Sequential Western blot analysis: After KIT knockdown in appropriate cell lines such as GIST T1, measure KCTD12 protein levels using anti-KCTD12 antibody (1:500-1:2000) . Previous studies showed both protein and mRNA expression levels of KCTD12 were significantly increased following KIT knockdown .

• Reciprocal analysis: Similarly, perform KCTD12 knockdown and assess KIT protein levels, although research suggests KCTD12 knockdown only slightly reduces KIT protein expression .

• Dose-dependent inhibition studies: Treat cells with increasing concentrations of imatinib (a KIT inhibitor) and measure KCTD12 protein levels to establish dose-response relationship.

• Time-course analysis: Monitor KCTD12 protein expression at different time points following KIT inhibition to determine the temporal dynamics of this regulatory relationship.

• Dual immunofluorescence: Perform co-staining with KCTD12 and KIT antibodies in tissue samples to assess inverse correlation of expression patterns.

• Chromatin immunoprecipitation (ChIP): If direct transcriptional regulation is suspected, use ChIP to investigate whether KIT-associated transcription factors bind to the KCTD12 promoter.

How should KCTD12 antibody staining be interpreted in colorectal cancer tissue microarrays for prognostic assessment?

For accurate interpretation of KCTD12 antibody staining in colorectal cancer tissue microarrays:

What are the optimal antibody-based methods to study KCTD12 protein domain functionality in relation to GABAB receptor signaling?

To study KCTD12 protein domain functionality using antibody-based approaches:

• Domain-specific antibodies: Use or develop antibodies that specifically recognize different domains of KCTD12, particularly the H1 domain which is crucial for GABAB receptor desensitization .

• Mutant protein detection: Generate and detect expression of KCTD12 domain mutants such as chimeric constructs (12-16H2) or truncation mutants (12-16H2Δ60, 12-16H2Δ113), which have been shown to have differential effects on GABAB receptor desensitization .

• Co-immunoprecipitation studies: Use KCTD12 antibodies for pull-down assays followed by detection of interacting partners such as GABAB2. Previous research has shown that chimeric proteins like 12-16H2, 12-Luc, and 12-Venus still co-immunoprecipitate with GABAB2 despite their inability to induce desensitization .

• Functional validation with electrophysiology: Correlate antibody detection of mutant expression with functional GABAB receptor desensitization measured through electrophysiological techniques like patch clamp recording of Kir3 currents .

• Subcellular localization analysis: Use immunofluorescence to track the localization of wild-type versus mutant KCTD12 proteins to determine if domain modifications affect trafficking or membrane association.

What are the recommended protocols for using KCTD12 antibody in detecting changes after drug treatments in cancer stemness models?

For optimal detection of KCTD12 changes following drug treatments in cancer stemness models:

• Antibody optimization for treatment conditions: When detecting KCTD12 after drug treatments such as 5-FU or imatinib, researchers should first validate antibody performance under treatment conditions as protein modifications may affect epitope accessibility. Previous studies showed HT29 cells with KCTD12 knockdown displayed enhanced viability in the presence of imatinib and 5-FU .

• Time-course experimental design: Collect samples at multiple time points (4h, 8h, 24h, 48h, 72h) post-treatment to capture dynamic changes in KCTD12 expression and downstream effects.

• Dual detection methodology:

  • Western blot (1:500-1:2000 dilution) for quantitative assessment of total KCTD12 protein levels

  • Immunofluorescence (1:10-1:100 dilution) for visualization of potential subcellular redistribution after treatment

• Sample preparation considerations: For cancer stemness models:

  • Spheroid cultures require careful disruption and fixation protocols

  • Side population cells identified by Hoechst-33342 dye should be isolated by FACS before antibody-based KCTD12 detection

• Parallel assessment of stemness markers: Simultaneously detect KCTD12 and stemness markers (CD44, CD133, CD29) to monitor the correlation between treatment effects on KCTD12 and subsequent changes in stemness .

How should KCTD12 antibody-based assays be designed when investigating potential tumor suppressor functions in different cancer types?

When designing antibody-based assays to investigate KCTD12's tumor suppressor functions:

• Tissue panel screening approach: Use KCTD12 antibody (1:50-1:500 for IHC) to screen expression across normal and malignant tissues from multiple cancer types . Previous studies documented decreased KCTD12 in CRC compared to normal tissue and pfetin (encoded by KCTD12) as a prognostic marker in GIST .

• Functional validation workflow:

  • Generate KCTD12 knockdown models using siRNA approaches similar to those employed in HT29 cells (for CRC) or GIST T1 cells

  • Confirm knockdown efficiency via Western blot (1:500-1:2000 dilution)

  • Assess proliferation, invasion, and drug resistance phenotypes

  • Previous studies showed KCTD12 knockdown increased cell proliferation in GIST T1 cells and enhanced chemoresistance in HT29 cells

• Mechanism investigation design:

  • Use co-immunoprecipitation with KCTD12 antibody to identify interacting partners

  • Perform pathway analysis focus on ERK signaling, which has been shown to be regulated by KCTD12 in CRC cells

  • Consider GABA B signaling pathway components, as KCTD12 has been reported to function via this pathway in some contexts

• Clinical correlation strategy:

  • Design tissue microarrays with adequate normal/tumor pairs and clinical follow-up data

  • Establish consistent scoring methods for KCTD12 IHC

  • Analyze correlation with established prognostic factors and patient outcomes