RBCK1 Antibody

What is RBCK1 and why is it relevant to biomedical research?

RBCK1 (RanBP-type and C3HC4-type zinc finger containing 1) functions primarily as an E3 ubiquitin-protein ligase that accepts ubiquitin from specific E2 ubiquitin-conjugating enzymes, such as UBE2L3/UBCM4, and subsequently transfers it to target substrates . This protein serves as a critical component of the LUBAC (linear ubiquitin chain assembly complex), which conjugates linear ('Met-1'-linked) polyubiquitin chains to various substrates and plays essential roles in NF-kappa-B activation and regulation of inflammation . RBCK1's significance extends beyond normal cellular functions to various pathological conditions including cardiomyopathy, renal cell carcinoma, and inflammatory disorders such as allergic rhinitis . The protein's molecular weight is approximately 58 kDa (calculated) with an observed weight of 57 kDa in experimental conditions, making it readily identifiable in various assay systems . Understanding RBCK1's roles in both physiological and pathological processes provides valuable insights into disease mechanisms and potential therapeutic targets, which explains its growing relevance in biomedical research contexts.

What types of RBCK1 antibodies are available for research applications?

Several types of RBCK1 antibodies have been developed for research purposes, each with specific characteristics suited for different experimental applications. Polyclonal antibodies, such as the rabbit polyclonal RBCK1 antibody (26367-1-AP) from Proteintech, offer broad epitope recognition which can enhance detection sensitivity in various applications . Monoclonal antibodies, including the rabbit recombinant monoclonal antibody [EPR28157-53] and mouse monoclonal antibody [CL4289] from Abcam, provide higher specificity for particular epitopes and greater consistency between batches . The host species for these antibodies includes rabbit and mouse, with each antibody being designed for specific recognition of human RBCK1, while some also demonstrate reactivity with mouse and rat RBCK1 samples . These antibodies vary in their immunogen design, with some targeting specific protein fragments (e.g., within amino acids 50-200 of human RBCK1) or fusion proteins (e.g., RBCK1 fusion protein Ag24405) . The diversity in antibody types allows researchers to select the most appropriate tool based on their specific experimental requirements, target species, and intended applications, thereby enhancing the reliability and reproducibility of RBCK1-related research.

How do researchers validate RBCK1 antibody specificity and functionality?

Validation of RBCK1 antibody specificity and functionality requires a multi-faceted approach to ensure reliable experimental results. Knockout (KO) and knockdown (KD) testing represents a gold standard for antibody validation, with several RBCK1 antibodies having documented validation through these methods as evidenced by multiple publications . Western blot analysis constitutes a fundamental validation technique, demonstrating the antibody's ability to recognize the target protein at the expected molecular weight (approximately 57 kDa for RBCK1), with nine publications supporting this application for certain RBCK1 antibodies . Immunohistochemistry validation involves testing on known positive tissues, such as human placenta tissue for RBCK1 antibodies, with specific antigen retrieval methods (TE buffer pH 9.0 or citrate buffer pH 6.0) being critical for optimal results . Cross-reactivity testing against various species samples helps determine the antibody's species specificity, with documented reactivity often including human samples as primary targets and, in some cases, mouse and rat samples . Additional validation approaches include immunofluorescence and immunoprecipitation, which further confirm the antibody's ability to recognize native RBCK1 in different experimental contexts . Thorough validation using these complementary techniques ensures that research findings based on RBCK1 antibody applications are reproducible and physiologically relevant.

What are the optimal protocols for using RBCK1 antibodies in immunohistochemistry?

Successful immunohistochemistry (IHC) with RBCK1 antibodies requires careful attention to protocol details to achieve specific and sensitive detection. Antigen retrieval represents a critical step when using RBCK1 antibodies for IHC, with recommended methods including TE buffer at pH 9.0, though citrate buffer at pH 6.0 may serve as an alternative depending on the specific tissue and fixation conditions . The optimal dilution range for RBCK1 antibodies in IHC applications typically falls between 1:50 and 1:500, though researchers should conduct titration experiments to determine the ideal concentration for their specific experimental system . Blocking protocols commonly employ bovine serum albumin (BSA) at room temperature for approximately 20 minutes before primary antibody application to minimize non-specific binding . Primary antibody incubation should proceed for 18 hours at 4°C for optimal results, followed by secondary antibody incubation at room temperature for 30 minutes . Visualization typically employs dextran polymer-conjugated horseradish-peroxidase and 3,3′-diaminobenzidine (DAB) chromogen, with hematoxylin counterstaining to provide cellular context . When evaluating RBCK1 expression by IHC, both staining intensity (scored from 0-3) and the proportion of positive cells should be considered to provide comprehensive expression analysis . Control slides lacking primary antibody should always be included to assess background staining and ensure specificity of the observed signals.

How should researchers design Western blot experiments using RBCK1 antibodies?

Western blot experiments using RBCK1 antibodies require careful experimental design and execution to ensure specific and reproducible detection. Sample preparation considerations should account for RBCK1's expected molecular weight of approximately 57-58 kDa, with gel percentage selection and running conditions optimized accordingly . Protein transfer parameters, including membrane type, transfer buffer composition, and transfer conditions (time, voltage, temperature), should be optimized to ensure efficient transfer of RBCK1 to the membrane for subsequent detection. Blocking solutions typically contain either non-fat dry milk or bovine serum albumin (BSA) to minimize non-specific binding, though specific recommendations may vary depending on the particular RBCK1 antibody being used. Primary antibody dilution must be carefully determined, with titration experiments recommended to establish optimal concentration for each specific experimental system and RBCK1 antibody lot . Secondary antibody selection should align with the host species of the primary RBCK1 antibody (e.g., anti-rabbit or anti-mouse), with appropriate dilution and incubation conditions. Positive and negative controls are essential for validating RBCK1 detection, with knockout or knockdown samples serving as valuable negative controls where available. Detection methods may include chemiluminescence, fluorescence, or colorimetric approaches, with sensitivity requirements dictating the optimal choice for a given experiment. Stripping and reprobing protocols may be necessary for additional protein detection on the same membrane, though care must be taken to avoid compromising RBCK1 signal integrity.

What considerations are important for immunoprecipitation experiments with RBCK1 antibodies?

Immunoprecipitation (IP) experiments with RBCK1 antibodies require specific technical considerations to successfully isolate RBCK1 and its interaction partners. Antibody selection is crucial, with documented IP-validated RBCK1 antibodies such as 26367-1-AP showing positive detection in human placenta tissue . Optimal antibody quantity for immunoprecipitation typically ranges from 0.5-4.0 μg of RBCK1 antibody for every 1.0-3.0 mg of total protein lysate, though titration experiments may be necessary to determine ideal concentrations for specific experimental conditions . Lysis buffer composition significantly impacts RBCK1 immunoprecipitation success, requiring careful consideration of detergent type and concentration, salt concentration, and pH to maintain RBCK1's native conformation and protein-protein interactions. Pre-clearing of lysates with appropriate control antibodies or protein A/G beads helps reduce non-specific binding and improves signal-to-noise ratio in subsequent analyses. Washing conditions following immunoprecipitation must balance removal of non-specifically bound proteins with preservation of genuine RBCK1 interactions, often requiring optimization of salt concentration and detergent levels in washing buffers. Detection methods for immunoprecipitated RBCK1 typically involve Western blotting with either the same or different RBCK1 antibodies, or mass spectrometry for interaction partner identification. Control experiments should include immunoprecipitation with isotype-matched non-specific antibodies and, where available, RBCK1 knockout or knockdown samples to confirm specificity of observed interactions.

What are common challenges when using RBCK1 antibodies and how can they be addressed?

Researchers frequently encounter several challenges when working with RBCK1 antibodies that require systematic troubleshooting approaches. Non-specific binding represents a common issue, particularly in Western blot and immunohistochemistry applications, which can be addressed by optimizing blocking conditions (using different blocking agents or concentrations), increasing antibody dilution, or employing more stringent washing procedures with higher detergent concentrations or salt content in washing buffers . Weak or absent signal may occur due to insufficient antigen retrieval in immunohistochemistry, which can be resolved by testing alternative antigen retrieval methods (comparing TE buffer pH 9.0 versus citrate buffer pH 6.0) or extending retrieval time and optimizing temperature conditions . Variable reactivity across different sample types can present challenges, particularly when comparing human versus mouse or rat samples, necessitating careful selection of antibodies with validated cross-reactivity for the species under investigation . Background issues in immunofluorescence applications may require optimization of fixation methods, permeabilization conditions, and careful titration of primary and secondary antibodies to improve signal-to-noise ratios. Inconsistent results between experimental replicates might stem from antibody degradation due to improper storage or handling, underscoring the importance of following recommended storage conditions (-20°C with glycerol and avoiding repeated freeze-thaw cycles) and considering aliquoting of antibody stocks for single-use applications .

How can researchers optimize dilution ratios for different RBCK1 antibody applications?

Optimizing dilution ratios for RBCK1 antibodies across different applications requires systematic titration experiments tailored to specific experimental conditions. For immunohistochemistry applications, the recommended dilution range for RBCK1 antibodies typically spans from 1:50 to 1:500, with researchers advised to conduct serial dilution experiments within this range to identify the optimal concentration that maximizes specific signal while minimizing background staining . Immunoprecipitation applications generally require 0.5-4.0 μg of RBCK1 antibody for 1.0-3.0 mg of total protein lysate, with preliminary experiments using varying antibody concentrations helping to establish the minimum effective concentration for successful target pull-down . Western blotting applications may require different dilution ratios depending on the specific RBCK1 antibody being used, sample type, and detection method, with titration experiments across a range of dilutions recommended for each new experimental system or antibody lot. Immunofluorescence applications typically require more concentrated antibody solutions compared to IHC or Western blotting, with optimization considering both signal intensity and background levels under specific microscopy settings. Flow cytometry applications necessitate careful titration to identify antibody concentrations that provide clear separation between positive and negative populations while maintaining acceptable signal-to-noise ratios. For all applications, researchers should consider that optimal dilution ratios may vary based on sample type (e.g., tissue sections versus cell lines), fixation method, and detection system sensitivity, underscoring the importance of sample-specific optimization rather than relying solely on manufacturer recommendations .

What validation methods confirm specific RBCK1 detection in experimental systems?

Comprehensive validation of RBCK1 antibodies requires multiple complementary approaches to ensure specificity and reliability of experimental results. Knockout/knockdown validation represents the gold standard, with several publications documenting RBCK1 antibody specificity using RBCK1 knockout or knockdown models, providing the most definitive evidence of antibody specificity . Western blot molecular weight verification involves confirming that the detected protein band appears at the expected molecular weight of 57-58 kDa for RBCK1, with additional validation possible through detection of expected size shifts in tagged constructs or truncated variants . Cross-reactivity testing against samples from multiple species helps establish species specificity, with documented reactivity of certain RBCK1 antibodies for human, mouse, and rat samples enabling appropriate experimental design and interpretation . Peptide competition assays, in which the antibody is pre-incubated with excess immunizing peptide before sample application, provide additional specificity confirmation when the expected signal is blocked or reduced. Correlation of protein expression with mRNA levels (e.g., comparing antibody staining with RNA-seq or qPCR data) provides orthogonal validation of expression patterns . Multiple antibody validation involves comparing results obtained with different RBCK1 antibodies targeting distinct epitopes, with concordant results providing strong evidence for specific detection. Positive control tissues with known RBCK1 expression, such as human placenta tissue, offer valuable reference points for validating new experimental systems or antibodies .

How does RBCK1 contribute to the regulation of NF-κB signaling and inflammation?

RBCK1 plays a sophisticated role in NF-κB signaling through its function as a component of the linear ubiquitin assembly complex (LUBAC), which conjugates linear ('Met-1'-linked) polyubiquitin chains to substrates critical for NF-κB activation . This E3 ligase complex, containing RBCK1, specifically targets IKBKG (also known as NEMO) and RIPK1 for linear ubiquitination, thereby facilitating the activation of canonical NF-κB and JNK signaling pathways that regulate inflammatory responses . The linear ubiquitination mediated by LUBAC and RBCK1 actively interferes with TNF-induced cell death processes, serving as a crucial regulatory mechanism that prevents excessive inflammation and maintains immune homeostasis . Upon TNF receptor stimulation, RBCK1-containing LUBAC is recruited to the TNF-R1 signaling complex (TNF-RSC) following initial polyubiquitination of TNF-RSC components by BIRC2 and/or BIRC3, where it then conjugates linear polyubiquitin to IKBKG and potentially other components, thus contributing to the stability and signaling capacity of this complex . Beyond its role in receptor-mediated signaling, RBCK1 and LUBAC contribute to innate immunity through the conjugation of linear polyubiquitin chains to the surface of bacteria invading the cytosol, forming part of the ubiquitin coat surrounding these pathogens, though interestingly, LUBAC cannot initiate this coat formation and can only extend pre-existing ubiquitin modifications . In allergic rhinitis, RBCK1 overexpression has been shown to attenuate inflammation by downregulating NLRP3, suggesting that RBCK1 may play context-dependent roles in different inflammatory conditions .

What is the mechanism of RBCK1-mediated p53 regulation in cancer development?

RBCK1 exerts significant influence on cancer development through its direct regulation of the tumor suppressor protein p53, particularly in renal cell carcinoma (RCC) where p53 mutations are relatively rare (approximately 2-3%) . The mechanistic basis of this regulation involves RBCK1 directly interacting with p53 protein, thereby facilitating its polyubiquitination and subsequent proteasomal degradation, which leads to suppression of p53 target genes and their associated tumor-suppressive functions . This interaction has profound consequences for cell proliferation, as evidenced by experiments demonstrating that RBCK1 depletion severely impacts the in vivo and in vitro proliferation of renal cancer cells, with these effects being rescuable through concurrent p53 knockdown in cell lines expressing wild-type p53 . RNA sequencing analysis of RCC cells following RBCK1 depletion has revealed that RBCK1 functions as a novel regulator of p53, influencing a network of p53-dependent genes involved in cell cycle arrest, apoptosis, and other critical cellular processes . The clinical relevance of this regulatory axis is underscored by findings that RBCK1 expression is upregulated in human RCC samples, with multiple public databases indicating a correlation between elevated RBCK1 expression and poor prognosis in RCC patients . The RBCK1-p53 regulatory relationship extends beyond RCC, with evidence suggesting similar mechanisms may operate in other cancer types, positioning RBCK1 as a potential therapeutic target for restoring p53 functions across multiple malignancies .

How do pathogenic RBCK1 variants contribute to cardiomyopathy and other disorders?

Pathogenic variants in the RBCK1 gene demonstrate significant implications for cardiac function, as exemplified by a recently discovered homozygous variant (c.598_599insT: p.His200LeufsTer14) in exon 8 that results in dilated cardiomyopathy (DCM) . This frameshift mutation leads to a premature termination codon, likely producing a truncated protein with compromised functionality or triggering nonsense-mediated mRNA decay, thereby disrupting critical RBCK1-mediated cellular processes in cardiac tissue . The inheritance pattern of RBCK1-related cardiomyopathy appears to follow autosomal recessive transmission, as evidenced by the identification of the variant in heterozygous form among all healthy family members of affected individuals, suggesting that a single functional copy of RBCK1 may be sufficient for normal cardiac development and function . Computational analyses using multiple predictive algorithms strongly support the pathogenicity of RBCK1 variants in cardiomyopathy, with tools such as MutationTaster, SIFT, and CADD scores providing in silico evidence for functional disruption . An intriguing pattern has emerged from literature review of RBCK1 variants, suggesting that the location of mutations within the protein structure correlates with specific phenotypic manifestations – variants affecting the N-terminal portion of RBCK1 appear more likely to manifest as immunodeficiency and auto-inflammation, while C-terminal modifications may preferentially impact cardiac function . The multi-system effects of RBCK1 variants highlight the protein's pleiotropic functions across different tissues and cellular contexts, with implications for diagnosis, genetic counseling, and potential therapeutic approaches for affected individuals .

What is the potential of RBCK1 as a therapeutic target in inflammatory diseases?

RBCK1 exhibits promising potential as a therapeutic target in inflammatory conditions, particularly in allergic rhinitis where RBCK1 overexpression has been demonstrated to attenuate inflammation through downregulation of NLRP3 . The mechanistic basis of this anti-inflammatory effect involves RBCK1-mediated suppression of the inflammatory and mobility progression in allergic rhinitis model cells, suggesting that therapeutic strategies aimed at enhancing RBCK1 expression or activity could provide clinical benefit in this common inflammatory condition affecting 10-30% of the global population . The relationship between RBCK1 and inflammatory mediators extends to its regulation of interleukin production, with experimental evidence showing that RBCK1 modulation affects levels of critical inflammatory cytokines including IL-18 and IL-33, which play central roles in allergic and inflammatory responses . RBCK1's role in the NF-κB signaling pathway provides additional therapeutic relevance, as NF-κB represents a master regulator of inflammation implicated in numerous inflammatory disorders, with RBCK1's position in the LUBAC complex offering potential for targeted intervention in this pathway . The cell-type specificity of RBCK1 expression in nasal epithelial cells suggests that targeted local delivery of RBCK1-modulating therapies might achieve anti-inflammatory effects while minimizing systemic adverse effects, an important consideration for chronic inflammatory conditions requiring long-term management . Therapeutic approaches might include small molecule enhancers of RBCK1 expression or activity, biologics targeting the RBCK1-NLRP3 axis, or potentially gene therapy approaches to increase local RBCK1 expression in affected tissues.

How can RBCK1 antibodies be utilized in cancer diagnosis and prognosis assessment?

RBCK1 antibodies offer significant utility in cancer diagnostics and prognostic evaluation through their ability to detect alterations in RBCK1 expression that correlate with disease progression and patient outcomes. Immunohistochemical analysis of tumor tissues using RBCK1 antibodies provides valuable diagnostic information, with upregulated RBCK1 expression having been documented in human renal cell carcinoma samples compared to adjacent normal tissues, suggesting potential use as a diagnostic biomarker . Prognostic applications of RBCK1 immunostaining are supported by analyses of multiple public databases revealing a correlation between elevated RBCK1 expression and poor prognosis in RCC patients, indicating that RBCK1 detection could serve as a stratification tool for identifying high-risk patients who might benefit from more aggressive therapeutic approaches . The specificity and sensitivity of RBCK1 as a cancer biomarker can be enhanced through standardized immunohistochemical protocols using validated antibodies, with scoring systems that account for both staining intensity (typically scored 0-3) and proportion of positive cells providing quantitative metrics for comparison across patient samples . Multi-marker diagnostic panels incorporating RBCK1 alongside other cancer-associated proteins may offer improved diagnostic accuracy compared to single-marker approaches, with antibody-based multiplexed immunohistochemistry or immunofluorescence techniques enabling simultaneous evaluation of multiple biomarkers in the same tissue section. Beyond solid tumors, RBCK1 antibodies may have applications in liquid biopsy approaches, potentially detecting circulating tumor cells or extracellular vesicles expressing RBCK1 as minimally invasive biomarkers for cancer detection and monitoring.

What role does RBCK1 play in the pathogenesis of cardiomyopathy and other genetic disorders?

RBCK1 plays a critical role in cardiac function and development, with pathogenic variants in the RBCK1 gene being implicated in the etiology of dilated cardiomyopathy (DCM) . The recent discovery of a homozygous likely pathogenic variant (c.598_599insT: p.His200LeufsTer14) in exon 8 of the RBCK1 gene in a 7-year-old girl with DCM provides direct evidence for RBCK1's causative role in this cardiac disorder, expanding the spectrum of genetic factors contributing to cardiomyopathies . Mechanistically, RBCK1 variants likely disrupt critical cellular processes including protein quality control, NF-κB signaling, and potentially mitochondrial function, all of which are essential for normal cardiac physiology and resilience to stress . The inheritance pattern of RBCK1-related cardiomyopathy appears to follow an autosomal recessive model, with healthy family members carrying heterozygous variants while affected individuals exhibit homozygous mutations, suggesting that a single functional copy of RBCK1 may be sufficient for normal cardiac development and function . Beyond cardiomyopathy, RBCK1 mutations have been associated with a spectrum of disorders involving immunodeficiency and auto-inflammation, with an emerging pattern suggesting that variants affecting different domains of the protein may result in distinct phenotypic manifestations – N-terminal protein modifications appearing more likely to cause immunodeficiency and inflammation, while C-terminal alterations may preferentially affect cardiac function . The pleiotropic effects of RBCK1 variants highlight the protein's diverse functional roles across multiple tissues and cellular processes, with implications for genetic counseling, clinical management, and potential therapeutic targeting in affected individuals .

What are the future research directions for RBCK1 antibody applications?

The evolving understanding of RBCK1's diverse biological functions opens numerous avenues for future research utilizing RBCK1 antibodies across multiple disciplines. Development of highly specific monoclonal antibodies targeting distinct RBCK1 domains could enable more precise mapping of protein interactions and functional regions, advancing our mechanistic understanding of RBCK1's roles in ubiquitination pathways and signaling cascades . Application of RBCK1 antibodies in high-throughput tissue microarray studies across diverse cancer types and stages would expand our knowledge of RBCK1's prognostic significance beyond renal cell carcinoma, potentially identifying additional malignancies where RBCK1 expression correlates with disease progression or therapeutic response . Integration of RBCK1 immunodetection with single-cell analysis technologies represents another promising direction, allowing characterization of RBCK1 expression heterogeneity within tissues and tumors at unprecedented resolution. Development of therapeutic antibodies targeting RBCK1 or its interaction partners could provide novel treatment approaches for conditions where RBCK1 dysregulation contributes to pathogenesis, such as certain cancers or inflammatory disorders . Investigation of RBCK1's roles in additional disease contexts, including neurological disorders, metabolic diseases, and aging-related conditions, may reveal previously unrecognized functions and therapeutic opportunities. Refinement of RBCK1 antibody-based diagnostic assays could improve early detection and risk stratification for RBCK1-associated disorders, including cardiomyopathies and cancers . These diverse research directions highlight the continuing importance of high-quality, well-validated RBCK1 antibodies for advancing both basic biological understanding and translational applications.