KLHL4 Antibody

What are the validated applications for KLHL4 antibodies?

KLHL4 antibodies have been extensively validated for multiple experimental applications critical for molecular and cellular biology research. The primary validated applications include Western Blotting (WB), Immunohistochemistry (IHC), Enzyme-Linked Immunosorbent Assay (ELISA), and Immunoprecipitation (IP) . These techniques allow researchers to detect, quantify, and characterize KLHL4 protein expression in different experimental contexts. Western Blotting typically requires dilutions between 1:500-1:2000 for optimal results, while IHC applications benefit from dilutions in the range of 1:20-1:200 . It is important to note that optimal dilutions may vary depending on the specific antibody clone, host species, and experimental conditions, necessitating preliminary optimization experiments for each new research application.

What antigen retrieval methods are recommended for KLHL4 immunohistochemistry?

Antigen retrieval is a critical step in immunohistochemistry (IHC) that significantly impacts KLHL4 antibody binding efficiency and signal intensity. For KLHL4 detection in formalin-fixed, paraffin-embedded (FFPE) tissues, the recommended antigen retrieval protocol involves using TE buffer at pH 9.0 . This alkaline pH buffer system effectively breaks protein cross-links formed during fixation, exposing KLHL4 epitopes for antibody binding. Alternatively, researchers may employ citrate buffer at pH 6.0 for antigen retrieval, though this may result in different staining intensity profiles . The choice between these buffer systems should be empirically determined based on the specific tissue type, fixation conditions, and the epitope targeted by the selected antibody. Optimization of heating time, temperature, and pressure conditions during antigen retrieval is also essential for balancing effective epitope unmasking with preservation of tissue morphology. Researchers should conduct preliminary experiments comparing different antigen retrieval methods when establishing KLHL4 IHC protocols for new tissue types or antibody clones.

What controls should be included when using KLHL4 antibodies in experimental procedures?

Rigorous experimental design for KLHL4 antibody applications requires appropriate positive and negative controls to ensure result validity and interpretability. For positive controls, researchers should use samples with confirmed KLHL4 expression, such as HeLa cells, which have been validated for KLHL4 detection by Western blotting . For tissue-based applications, human brain tissue has been confirmed as appropriate positive control material for KLHL4 immunohistochemistry . Negative controls should include isotype-matched immunoglobulins lacking KLHL4 specificity to assess non-specific binding. Additionally, when available, blocking peptides specific to the KLHL4 antibody epitope, such as catalog number 33R-1743, provide valuable specificity controls . For genetic validation approaches, KLHL4 knockout cell lines (such as Klhl4 knocked-out MEF cells) can serve as definitive negative controls . The inclusion of loading controls (e.g., housekeeping proteins) for Western blot applications and tissue-specific internal controls for IHC is also essential for accurate data interpretation and normalization across experimental conditions.

How can KLHL4 antibodies be utilized to investigate p53-dependent pathways?

KLHL4 antibodies offer powerful tools for exploring p53-mediated cellular pathways, as KLHL4 has been identified as a novel p53 target gene. Research has demonstrated that KLHL4 mRNA and protein expression significantly increase (approximately 3-fold) in response to ectopic p53 expression or DNA damage . Researchers investigating p53-dependent stress responses can employ KLHL4 antibodies in combination with p53-activating treatments (e.g., DNA damaging agents, Nutlin-3a) to monitor KLHL4 induction as a downstream marker of p53 activation. Co-immunoprecipitation experiments using KLHL4 and p53 antibodies can verify the physical interaction between these proteins and identify additional complex components. Furthermore, chromatin immunoprecipitation (ChIP) assays using KLHL4 antibodies can investigate how KLHL4 contributes to p53 binding at the p21 promoter, as KLHL4 has been shown to enhance p53-mediated p21 transcription . This multi-faceted approach using KLHL4 antibodies enables comprehensive characterization of this newly recognized component of the p53 tumor suppressor network.

What methodological approaches can address the dual role of KLHL4 in p21 regulation and Cul3-mediated ubiquitination?

Recent discoveries regarding KLHL4's dual functionality in p21 regulation and protein ubiquitination necessitate sophisticated experimental designs employing KLHL4 antibodies. To investigate KLHL4's role in p21 transcriptional activation, researchers should combine chromatin immunoprecipitation (ChIP) using KLHL4 antibodies with reporter gene assays containing the p21 promoter region . Quantitative immunoblotting with KLHL4 and p21 antibodies following genetic manipulation of KLHL4 expression (overexpression, knockdown, or knockout) can establish causal relationships between these proteins. For examining KLHL4's interaction with the Cul3 ubiquitin ligase complex, co-immunoprecipitation experiments using KLHL4 antibodies followed by Cul3 detection can confirm complex formation . Ubiquitination assays incorporating KLHL4 antibodies for protein immunoprecipitation, followed by ubiquitin detection, can identify KLHL4-dependent ubiquitination targets. Cell proliferation assays comparing wild-type and KLHL4-deficient cells (such as the Klhl4 knocked-out MEF model) can further validate KLHL4's functional impact on cellular growth control mechanisms . This comprehensive methodology allows researchers to dissect the mechanistic details of KLHL4's multifunctional roles in cellular homeostasis.

How can epitope-specific KLHL4 antibodies be leveraged to distinguish protein domains and functional regions?

The availability of KLHL4 antibodies targeting different epitopes provides unique opportunities for domain-specific functional analysis of this complex protein. Researchers can strategically select antibodies recognizing distinct regions, such as N-terminal (amino acids 1-100), mid-region (amino acids 125-175), or specific epitopes (amino acids 35-84, 84-113) , to investigate domain-specific functions. Comparative immunoprecipitation experiments using different epitope-specific antibodies can reveal domain-dependent protein interactions, potentially identifying partners that associate with specific KLHL4 structural elements. For proteins containing both BTB/POZ domains and kelch repeats like KLHL4, domain-specific antibodies can help determine which region mediates Cul3 binding versus substrate recognition. Additionally, researchers can employ epitope-specific antibodies in immunofluorescence experiments to track potential differential subcellular localization of KLHL4 domains. When combined with truncation or domain mutation constructs, epitope-specific antibodies enable precise mapping of functional regions critical for KLHL4's roles in p53 cooperation, p21 regulation, and ubiquitin-dependent proteolysis pathways.

What are the common challenges in KLHL4 detection and how can they be addressed?

Researchers frequently encounter several technical challenges when working with KLHL4 antibodies. One common issue is variable signal intensity across different cell types or tissues, which stems from naturally different KLHL4 expression levels and potential epitope masking. To address this, researchers should optimize protein extraction conditions, considering specialized lysis buffers that effectively solubilize membrane-associated proteins while preserving epitope integrity. For Western blot applications, transfer efficiency can significantly impact KLHL4 detection; larger proteins may require extended transfer times or specialized buffer systems. Signal amplification strategies, such as using polymer-based detection systems for IHC or enhanced chemiluminescence substrates for WB, can improve detection sensitivity. Non-specific binding, another common challenge, can be minimized by thorough blocking procedures and careful antibody dilution optimization. Additionally, researchers should be aware that p53 status in experimental models might influence KLHL4 expression levels , potentially affecting detection sensitivity. Incorporating proper controls and standardizing experimental conditions across studies is essential for generating reproducible and reliable KLHL4 detection results.

How can researchers validate KLHL4 antibody specificity for their particular experimental system?

Thorough validation of KLHL4 antibody specificity is essential for generating reliable research data. A comprehensive validation approach should begin with testing multiple antibody dilutions (e.g., 1:500-1:2000 for WB, 1:20-1:200 for IHC) to determine optimal signal-to-noise ratios. Researchers should compare KLHL4 detection patterns across multiple antibodies targeting different epitopes to confirm consistent protein identification. When available, genetic approaches using KLHL4 knockdown (siRNA, shRNA) or knockout (CRISPR-Cas9) systems provide definitive specificity controls by demonstrating signal reduction or elimination. Peptide competition assays using specific blocking peptides, such as catalog number 33R-1743 , can verify epitope-specific binding. Mass spectrometry analysis of immunoprecipitated proteins can provide unbiased confirmation of antibody target identity. For tissue applications, researchers should compare KLHL4 antibody staining patterns with known KLHL4 expression profiles from transcriptomic databases. This multi-faceted validation approach ensures that experimental findings genuinely reflect KLHL4 biology rather than antibody artifacts, particularly important for this relatively understudied protein where literature precedent may be limited.

What methodological modifications are necessary when studying KLHL4 in different experimental models?

Adapting KLHL4 antibody protocols for diverse experimental models requires systematic optimization and model-specific considerations. For cell culture models, researchers should account for potential differences in KLHL4 expression levels between cell types, adjusting antibody concentrations accordingly. When transitioning between adherent and suspension cells, protein extraction protocols may require modification to ensure comparable KLHL4 recovery. For tissue-based applications, optimal fixation conditions vary by tissue type; generally, 10% neutral buffered formalin with controlled fixation duration minimizes epitope masking while preserving tissue architecture. Species differences necessitate careful antibody selection, as not all KLHL4 antibodies demonstrate cross-reactivity with mouse, rat, or zebrafish samples . When working with developmental models, researchers should consider potential temporal regulation of KLHL4 expression, adjusting detection sensitivity accordingly. For in vivo imaging applications, fluorophore-conjugated secondary antibodies must be carefully selected to avoid tissue autofluorescence interference. Importantly, given KLHL4's regulation by p53 , researchers should carefully document the p53 status of their experimental models, as this may significantly impact baseline KLHL4 expression levels and experimental outcomes.

What are the emerging research applications for KLHL4 antibodies in cancer and developmental biology?

KLHL4 antibodies are poised to make significant contributions to both cancer research and developmental biology. Given KLHL4's identification as a p53 target gene that inhibits cell proliferation , these antibodies will be invaluable for investigating KLHL4's potential tumor suppressor functions across diverse cancer types. Immunohistochemistry with KLHL4 antibodies could reveal expression patterns in tumor tissue microarrays, potentially identifying correlations with patient outcomes or treatment responses. In developmental biology, KLHL4's association with X-linked cleft palate (CPX) suggests critical roles in craniofacial development that could be visualized through immunohistochemical studies at different embryonic stages. Researchers can employ KLHL4 antibodies to track protein expression dynamics during cellular differentiation processes, potentially uncovering novel developmental functions beyond craniofacial morphogenesis. The availability of KLHL4 antibodies with zebrafish reactivity further enhances opportunities for developmental studies in this versatile model organism. As research progresses, KLHL4 antibodies may facilitate the identification of novel biomarkers, therapeutic targets, or developmental regulatory mechanisms with clinical significance.

How can researchers integrate KLHL4 antibody-based approaches with modern multi-omics technologies?

The integration of KLHL4 antibody-based methodologies with cutting-edge multi-omics technologies offers unprecedented opportunities for comprehensive protein characterization. Researchers can combine KLHL4 immunoprecipitation with mass spectrometry (IP-MS) to identify novel interacting partners within the p53 pathway or Cul3 ubiquitination network. ChIP-sequencing using KLHL4 antibodies can map genome-wide binding patterns, potentially revealing roles beyond p21 regulation . Correlation of KLHL4 protein levels (detected via antibody-based proteomics) with transcriptomic data could identify discrepancies suggesting post-transcriptional regulation mechanisms. For spatial context, combining KLHL4 immunohistochemistry with spatial transcriptomics can reveal tissue microenvironments where KLHL4 function is particularly critical. Proximity labeling approaches (BioID, APEX) coupled with KLHL4 antibody validation can identify transient or context-specific protein interactions. Additionally, single-cell proteomics incorporating KLHL4 antibodies could reveal cell-type-specific expression patterns lost in bulk analysis. These integrated approaches leverage the specificity of KLHL4 antibodies while exploiting the comprehensive coverage of omics technologies, potentially accelerating discoveries regarding KLHL4's roles in development, disease, and cellular homeostasis.