KLHDC4 Antibody

What is KLHDC4 and what is its biological significance?

KLHDC4 (Kelch Domain Containing 4) is a 520 amino acid protein characterized by the presence of six kelch repeats. It exists in three alternatively spliced isoforms that may play distinct roles in cellular processes. KLHDC4 is primarily localized in the cytoplasm, where it is involved in various signaling pathways and cellular functions, including regulation of actin cytoskeleton dynamics and protein degradation .

The protein's interactions with other cytoplasmic proteins influence cellular responses to stress and maintain cellular homeostasis. The gene encoding KLHDC4 is situated on human chromosome 16q24.2, a region associated with several genetic disorders, including Crohn's disease and Rubinstein-Taybi syndrome . Recent research has identified KLHDC4 as a potential marker for cancer progression, particularly in nasopharyngeal carcinoma (NPC) .

Evaluating antibody specificity is crucial for reliable research outcomes. For KLHDC4 antibodies, consider these validation approaches:

• Knockout validation: Using CRISPR/Cas9-mediated KLHDC4 knockout cell lines as negative controls. This approach involves transfecting cells with KLHDC4 sgRNA and validating knockout clones by western blotting and Sanger sequencing .

• Multiple antibody approach: Using antibodies from different sources that recognize distinct epitopes of KLHDC4 to confirm consistent results.

• Peptide competition assay: Pre-incubating the antibody with the immunizing peptide should eliminate specific binding in immunoblotting or immunostaining.

• Cross-species reactivity assessment: Verifying whether the antibody recognizes KLHDC4 in multiple species (human, mouse, rat) as claimed by manufacturers .

• Protein array screening: Some antibodies, such as Prestige Antibodies, are tested against protein arrays of 364 human recombinant protein fragments to ensure specificity .

What are the optimal protocols for immunohistochemical detection of KLHDC4?

For optimal immunohistochemical detection of KLHDC4 in tissue samples:

• Sample preparation: Fix tissues in 10% formalin and embed in paraffin. Deparaffinize and rehydrate sections before staining .

• Antigen retrieval: Retrieve antigens using appropriate buffer systems (specific protocols may vary by antibody).

• Blocking and primary antibody incubation: Block endogenous peroxidase activity with 1% H₂O₂ in PBS for 30 minutes. Incubate slides with anti-KLHDC4 primary antibody at 4°C overnight .

• Detection system: Use Envision System with diaminobenzidine (Dako) for visualization .

• Scoring: Implement a semi-quantitative scoring criterion that accounts for both staining intensity (negative: 0; weak: 1; moderate: 2; strong: 3) and proportion of immunopositive cells (<25%: 1; 25-50%: 2; 50-75%: 3; ≥75%: 4). Calculate staining index (values 0-7) by combining these scores .

• Categorization: Subdivide scores according to a cutoff value of the ROC curve into low expression (≤4.5) and high expression (>4.5) .

This approach has been validated in studies examining KLHDC4 expression in nasopharyngeal carcinoma tissues, showing significant associations with clinical parameters .

How can researchers optimize Western blot detection of KLHDC4?

For optimal Western blot detection of KLHDC4:

• Sample preparation: Extract proteins from cells or tissues using appropriate lysis buffers containing protease inhibitors.

• Antibody selection and dilution: For polyclonal antibodies like those from Abbexa, use approximately 1:1000 dilution . For monoclonal antibodies like D-10, follow manufacturer recommendations.

• Molecular weight verification: Look for bands at approximately 57.9 kDa, which is the calculated molecular weight of KLHDC4 . Be aware that alternative splicing may result in multiple isoforms with different molecular weights.

• Positive controls: Include lysates from cell lines known to express KLHDC4, such as CNE2, SUNE1, or other NPC cell lines that have shown high expression .

• Negative controls: If available, use KLHDC4 knockout cell lysates as the most stringent negative control .

• Loading controls: Include appropriate housekeeping proteins for normalization.

• Signal enhancement: For challenging detections, consider using antibody bundles that include signal enhancers, such as m-IgG Fc BP-HRP or m-IgGκ BP-HRP bundled products .

What approaches can be used to study KLHDC4 localization in cells?

KLHDC4 is primarily localized in the cytoplasm, and the following methods can be used to study its subcellular distribution:

• Immunofluorescence (IF): Several anti-KLHDC4 antibodies are validated for IF applications . Use appropriate fixation (4% paraformaldehyde) and permeabilization (0.1% Triton X-100) protocols.

• Subcellular fractionation: Separate cytoplasmic, membrane, nuclear, and cytoskeletal fractions and analyze KLHDC4 distribution by Western blotting.

• Co-localization studies: Perform dual immunostaining with markers for specific organelles or cytoskeletal structures to determine precise localization patterns.

• Live-cell imaging: Consider expressing KLHDC4-GFP fusion proteins to track localization in living cells.

• High-resolution microscopy: For detailed localization studies, use super-resolution microscopy techniques such as STED or STORM.

When performing these experiments, it's important to note that different KLHDC4 isoforms may show distinct localization patterns, and overexpression systems might not perfectly recapitulate endogenous localization .

How can researchers establish and validate KLHDC4 knockout models?

CRISPR/Cas9-mediated KLHDC4 knockout models have been successfully established using the following methodology:

• sgRNA design: Select guide RNAs targeting exon 5 of the KLHDC4 gene, which has been shown to be effective .

• Transfection approach: Co-transfect cells with 1 μg of KLHDC4 sgRNA#2 plasmid plus 1 μg of pSpCas9(BB)-2A-GFP plasmid using Lipofectamine 2000 .

• Cell sorting and clonal isolation: Use GFP as a fluorescent marker to sort transfected cells 48 hours post-transfection via fluorescence-activated cell sorting (FACS) into 96-well plates to isolate single cells .

• Knockout validation: Validate knockout clones through:

  • Western blotting to confirm absence of KLHDC4 protein

  • Sanger sequencing to confirm genetic modifications

  • Functional assays to assess phenotypic changes

This approach has been successfully implemented in CNE2 NPC cell lines, demonstrating that KLHDC4 knockout significantly inhibits cell proliferation, colony formation, and tumor formation, while inducing apoptosis .

What is the relationship between KLHDC4 expression and cancer prognosis?

Research has established significant associations between KLHDC4 expression and cancer outcomes, particularly in nasopharyngeal carcinoma:

These findings suggest that KLHDC4 antibodies are valuable tools for assessing prognosis in NPC patients and potentially in other cancer types where KLHDC4 may play similar roles.

How do different epitope targets affect KLHDC4 antibody performance?

The choice of epitope can significantly impact antibody performance across different applications:

When selecting an antibody based on epitope, researchers should consider:

• The specific isoforms they aim to detect

• Whether post-translational modifications might affect epitope accessibility

• The native versus denatured state of the protein in their application

• Whether the epitope is conserved across species (for cross-species studies)

What are common challenges in KLHDC4 immunodetection and how can they be addressed?

Researchers may encounter several challenges when working with KLHDC4 antibodies:

• Multiple bands in Western blotting:

  • Cause: KLHDC4 exists in three alternatively spliced isoforms

  • Solution: Use antibodies that target conserved regions or run positive controls with recombinant KLHDC4 proteins of known sizes

• Weak signal in immunostaining:

  • Cause: Low endogenous expression in certain tissues

  • Solution: Optimize antigen retrieval methods, increase antibody concentration, or use signal amplification systems

• Non-specific binding:

  • Cause: Cross-reactivity with related kelch domain-containing proteins

  • Solution: Increase blocking time/concentration, optimize antibody dilution, or consider using KLHDC4 knockout cells as negative controls

• Variability between different lots of antibodies:

  • Cause: Batch-to-batch variation in production

  • Solution: Standardize validation protocols and maintain reference samples for comparison

• Species cross-reactivity issues:

  • Cause: Sequence differences between species

  • Solution: Verify whether the antibody is validated for your species of interest and check sequence homology in the epitope region

What controls should be included in KLHDC4 antibody-based experiments?

To ensure reliable results in KLHDC4 antibody experiments, include these controls:

• Positive tissue/cell controls:

  • NPC cell lines (CNE2, SUNE1, SUNE2, HK1, HNE1, HONE1) that express high levels of KLHDC4

  • Nasopharyngeal carcinoma tissue sections with confirmed KLHDC4 expression

• Negative controls:

  • Immortalized normal nasopharyngeal epithelial cell lines (NP69, NPEC-N2-Bmi1, NPEC-N5-Tert) with low KLHDC4 expression

  • KLHDC4 knockout cell lines generated via CRISPR/Cas9

  • Primary antibody omission controls

  • Isotype controls (especially for monoclonal antibodies)

• Antibody validation controls:

  • Peptide competition assays using the immunizing peptide

  • Multiple antibodies targeting different epitopes

  • Recombinant KLHDC4 protein standards

• Technical controls:

  • Loading controls for Western blotting (β-actin, GAPDH)

  • Internal reference controls for immunostaining

  • Quantitative standards for ELISA applications

Including these controls allows researchers to distinguish specific signals from background and validate the reliability of their observations across different experimental conditions.

How can researchers interpret contradictory results from different KLHDC4 antibodies?

When faced with contradictory results from different KLHDC4 antibodies, consider these methodological approaches:

• Epitope mapping comparison:

  • Compare the epitope regions recognized by each antibody

  • Determine if discrepancies could be due to detection of different isoforms

  • Check if post-translational modifications might affect epitope accessibility

• Validation stringency assessment:

  • Review validation data for each antibody (Western blot, IHC, knockout validation)

  • Evaluate the robustness of controls used in the validation process

  • Consider the extent of validation across different applications

• Experimental conditions analysis:

  • Compare protocols used (fixation methods, antigen retrieval, blocking conditions)

  • Assess differences in detection systems (direct vs. indirect, amplification methods)

  • Evaluate sample preparation differences

• Combinatorial approach:

  • Use multiple antibodies in parallel on the same samples

  • Employ orthogonal detection methods (e.g., mass spectrometry) for verification

  • Consider genetic approaches (siRNA, CRISPR) to validate specificity

• Literature cross-reference:

  • Review published data on KLHDC4 expression and localization

  • Contact antibody manufacturers for additional validation data

  • Consult with researchers who have published using these antibodies

When reporting contradictory results, document all methodological details and provide transparent discussion of possible explanations for the discrepancies observed.

How might KLHDC4 antibodies be utilized in clinical applications?

Based on current research findings, KLHDC4 antibodies show potential for clinical applications:

• Prognostic biomarker development:

  • KLHDC4 expression correlates with poor prognosis in NPC patients

  • IHC scoring systems have been developed that could be standardized for clinical use

  • Multi-center validation studies could establish KLHDC4 as a routine prognostic marker

• Therapeutic target assessment:

  • Antibodies can be used to screen for KLHDC4 expression before implementing targeted therapies

  • Monitoring KLHDC4 levels during treatment could help assess therapy efficacy

  • Development of KLHDC4-targeting therapeutics would require reliable detection methods

• Companion diagnostics:

  • If KLHDC4-targeted therapeutics are developed, validated antibodies would be essential as companion diagnostics

  • Standardized IHC protocols could help identify patients likely to respond to such therapies

• Early detection strategies:

  • Further validation could determine if KLHDC4 overexpression occurs early in carcinogenesis

  • Sensitive detection methods might enable earlier diagnosis of high-risk lesions

Significant validation studies and regulatory approval processes would be required before clinical implementation, but the groundwork for such applications is being laid by current research.

What are emerging technologies for studying KLHDC4 protein interactions?

Advanced methodologies for investigating KLHDC4 protein interactions include:

• Proximity labeling approaches:

  • BioID or TurboID fusion proteins to identify proteins in close proximity to KLHDC4 in living cells

  • APEX2-based proximity labeling for temporal control of labeling reactions

• Advanced co-immunoprecipitation techniques:

  • Quantitative immunoprecipitation combined with knockout (QUICK) to identify specific interactors

  • Cross-linking immunoprecipitation (CLIP) to capture transient interactions

• Protein complementation assays:

  • Split fluorescent protein systems to visualize KLHDC4 interactions in living cells

  • NanoBiT or NanoLuc complementation for sensitive detection of protein-protein interactions

• Mass spectrometry-based approaches:

  • Thermal proteome profiling to identify binding partners

  • Cross-linking mass spectrometry to map interaction interfaces

  • SILAC-based quantitative proteomics to compare interactomes under different conditions

• Single-molecule techniques:

  • Single-molecule FRET to study conformational changes upon binding

  • Super-resolution microscopy to visualize interaction dynamics in cells

These emerging techniques could help elucidate KLHDC4's role in signaling pathways, actin cytoskeleton dynamics, and protein degradation, potentially revealing new therapeutic targets.