RUVBL1 Antibody

What is RUVBL1 and what cellular functions does it regulate?

RUVBL1 (also known as Pontin, TIP49, NMP238) is a member of the AAA+ (ATPase associated with diverse cellular activities) protein family that consists of 456 amino acids with a molecular weight of approximately 50 kDa . It associates with numerous chromatin-remodeling complexes and plays important roles in transcriptional regulation, DNA damage response, telomerase activity, snoRNP assembly, cellular transformation, and cancer metastasis . RUVBL1 interacts with TATA-binding protein (TBP), which is central to transcriptional regulation through complex formation with various transcription regulators . Additionally, RUVBL1 functions as a component of a large nuclear protein complex, potentially the RNA polymerase II holoenzyme, and shares high homology with RuvB proteins that function as DNA helicases promoting branch migration of Holliday junctions .

What are the most effective applications for RUVBL1 antibody detection?

RUVBL1 antibodies demonstrate effectiveness across multiple experimental applications, with varying sensitivity depending on the specific antibody clone and sample type. Western blot analysis shows consistently robust results across different cell lines, including U2OS, HeLa, HT-1080, HCT 116, K-562, A549, and A431 cells with recommended dilutions ranging from 1:1000 to 1:50000 . Immunohistochemistry (IHC) can be successfully performed on tissues such as mouse liver and human ovarian tumors using dilutions of 1:50-1:500, with antigen retrieval using TE buffer pH 9.0 or citrate buffer pH 6.0 . Immunoprecipitation (IP) has been validated in HeLa cells, requiring 0.5-4.0 μg of antibody per 1.0-3.0 mg of total protein lysate . Immunofluorescence (IF) applications work effectively in cell lines such as A431 at dilutions of 1:50-1:500 . For optimal results, researchers should titrate the antibody concentration within the recommended ranges for each specific experimental system .

How should RUVBL1 antibodies be stored and handled to maintain reactivity?

RUVBL1 antibodies require specific storage conditions to preserve their reactivity and stability. Commercial RUVBL1 antibodies are typically supplied in PBS buffer containing 0.02% sodium azide and 50% glycerol at pH 7.3 . They should be stored at -20°C, where they remain stable for approximately one year after shipment . For the -20°C storage, aliquoting is generally unnecessary, which minimizes freeze-thaw cycles that can degrade antibody quality . Some antibody preparations may contain 0.1% BSA as a stabilizer, particularly in smaller volume formats (20 μl) . It's important to note that although the antibodies are stable at -20°C, prolonged storage at 4°C or room temperature should be avoided as this can lead to loss of activity. When working with the antibodies, minimize repeated freeze-thaw cycles and keep them on ice during experimental procedures to maintain optimal reactivity .

What control samples should be included when validating RUVBL1 antibody specificity?

When validating RUVBL1 antibody specificity, researchers should implement a multi-tiered control strategy. Positive controls should include cell lines with confirmed RUVBL1 expression such as U2OS, HeLa, HT-1080, HCT-116, K-562, A549, and A431 cells, which consistently show robust RUVBL1 detection by Western blot . Negative controls should incorporate RUVBL1 knockdown or knockout models, as numerous publications have utilized RUVBL1 KD/KO systems for antibody validation . For antibody specificity testing, pre-absorption controls using the immunogen peptide can help confirm binding specificity. Additionally, comparative analysis using multiple antibodies targeting different epitopes of RUVBL1 can enhance confidence in specificity. When performing tissue-based experiments, normal adjacent tissue serves as an appropriate comparative control for expression levels. Finally, for assessing cross-reactivity, researchers should note that most commercially available RUVBL1 antibodies have been validated for reactivity with human and mouse/rat samples, though cross-reactivity with other species should be empirically determined .

How can RUVBL1 antibodies be used to investigate its role in DNA damage repair mechanisms?

Investigation of RUVBL1's role in DNA damage repair requires specialized methodological approaches using validated antibodies. Recent research has demonstrated that RUVBL1 enhances DNA damage repair and confers radioresistance in breast cancer cells both in vitro and in vivo . To study these mechanisms, researchers can utilize RUVBL1 antibodies in chromatin immunoprecipitation (ChIP) assays to assess RUVBL1 recruitment to DNA damage sites following radiation exposure . Co-immunoprecipitation (Co-IP) experiments using RUVBL1 antibodies can identify interaction partners within repair complexes, particularly the RUVBL1/2-β-catenin transcription complex that forms after radiation treatment . Immunofluorescence microscopy with RUVBL1 antibodies can visualize subcellular relocalization during the DNA damage response, especially following irradiation . Western blotting should be performed to quantify RUVBL1 expression changes after DNA damage induction, as protein profiling has shown increased RUVBL1 levels after irradiation in mouse breast cancer models . Additionally, researchers can combine RUVBL1 antibody-based detection with H4K16 acetylation markers to investigate how RUVBL1 influences histone modifications during homologous recombination repair and promotes expression of non-homologous end joining (NHEJ) repair-related proteins .

What are the technical considerations when detecting RUVBL1-RUVBL2 complexes in cellular systems?

Detection of RUVBL1-RUVBL2 complexes presents several technical challenges requiring careful experimental design. These proteins typically form a complex with a distinctive molecular signature appearing as doublets of approximately 50 kDa when visualized by protein immunoprecipitation assays . For optimal complex detection, sequential immunoprecipitation (sequential IP) is recommended, first pulling down with RUVBL1 antibody followed by RUVBL2 antibody, or vice versa, to ensure isolation of the complete complex . Native gel electrophoresis or blue native PAGE should be used instead of denaturing SDS-PAGE when preserving the intact complex is necessary . Researchers should be aware that standard lysis buffers may disrupt the complex; therefore, gentle lysis conditions using buffers containing low concentrations of non-ionic detergents are preferable . When performing co-immunoprecipitation experiments, cross-linking agents such as DSP (dithiobis[succinimidylpropionate]) can stabilize transient protein-protein interactions within the complex . For analyzing complex formation under physiological conditions, proximity ligation assays (PLA) using antibodies against both RUVBL1 and RUVBL2 can provide spatial information about complex formation in situ . It's important to note that the RUVBL1/2 complex can associate with various other proteins in different cellular contexts, so experimental conditions may need to be optimized for the specific cellular system being investigated .

How does ubiquitination affect RUVBL1 detection and what methodologies can overcome these challenges?

Ubiquitination of RUVBL1 presents significant challenges for antibody-based detection that require specialized approaches. Research has shown that DTL (Denticleless protein homolog) modifies RUVBL1 through K63 ubiquitination during radiation treatment, which promotes formation of the RUVBL1/2-β-catenin transcription complex . To effectively detect ubiquitinated RUVBL1, researchers should first perform immunoprecipitation under denaturing conditions (1-2% SDS, boiled, then diluted) to disrupt protein-protein interactions while preserving the ubiquitin modifications . A dual-immunoprecipitation approach can be employed—first pulling down with ubiquitin antibodies followed by RUVBL1 detection, or vice versa—to specifically isolate ubiquitinated RUVBL1 . When performing Western blot analysis, higher percentage gels (10-12%) with extended run times improve separation of ubiquitinated RUVBL1 species, which appear as higher molecular weight bands above the standard 50 kDa RUVBL1 protein . Researchers should also consider using deubiquitinase inhibitors (like N-ethylmaleimide or PR-619) in lysis buffers to preserve ubiquitination modifications during sample preparation . For distinguishing between different ubiquitin chain types, particularly K63-linked chains that affect RUVBL1 function, linkage-specific ubiquitin antibodies should be used in combination with RUVBL1 antibodies . Mass spectrometry analysis following RUVBL1 immunoprecipitation provides the most comprehensive approach for identifying ubiquitination sites and quantifying modification levels across different experimental conditions .

What are the critical differences in detecting RUVBL1 in normal versus cancer tissues?

Detecting RUVBL1 in normal versus cancer tissues requires awareness of several critical differences that influence experimental design and interpretation. In cancer contexts, particularly breast cancer, RUVBL1 expression levels are significantly upregulated following radiation treatment, requiring calibrated antibody dilutions to prevent signal saturation in comparative studies . Subcellular localization patterns differ between normal and cancer tissues—while RUVBL1 typically shows nuclear localization in normal cells, cancer tissues may exhibit both nuclear and cytoplasmic distribution, necessitating careful immunohistochemical or immunofluorescence analysis with appropriate nuclear/cytoplasmic markers . The RUVBL1/2 complex forms distinct functional assemblies in cancer cells, particularly after radiation treatment, where it associates with β-catenin to form transcription complexes that promote radioresistance . For cancer tissue analysis, antigen retrieval methods may require optimization beyond the standard protocols used for normal tissues, with TE buffer pH 9.0 often providing better results than citrate buffer pH 6.0 . Post-translational modifications, especially ubiquitination by DTL, are more prevalent in cancer tissues and affect antibody binding efficiency, potentially requiring antibodies that recognize specific regions of RUVBL1 unaffected by these modifications . When performing quantitative comparisons, researchers should normalize RUVBL1 expression to multiple housekeeping proteins rather than a single reference, as conventional housekeeping genes may be differentially expressed in cancer versus normal tissues .

How should researchers optimize Western blot protocols for RUVBL1 detection across different cellular systems?

Optimizing Western blot protocols for RUVBL1 detection requires systematic adjustment of multiple parameters based on the cellular system being studied. RUVBL1 appears as a distinct band at approximately 50 kDa, consistent with its calculated molecular weight of 456 amino acids . For cell lysis, RIPA buffer supplemented with protease inhibitors provides efficient extraction while maintaining protein integrity, though gentler lysis buffers may be preferred when studying RUVBL1 complexes . Protein loading should be standardized between 20-40 μg per lane, with higher amounts potentially necessary for cells with lower RUVBL1 expression . Gel percentage is critical—10-12% polyacrylamide gels offer optimal separation of the 50 kDa RUVBL1 protein from potential post-translationally modified variants .

Transfer conditions should be optimized for proteins in the 50 kDa range, typically using semi-dry transfer at 15-20V for 30-45 minutes or wet transfer at 100V for 60-90 minutes . Blocking solutions vary in effectiveness by cell type—5% non-fat dry milk in TBST works well for most applications, though 3-5% BSA may provide lower background in some systems . Antibody dilutions should be titrated for each cellular system, with recommended ranges between 1:1000-1:10000 for polyclonal antibodies and 1:5000-1:50000 for monoclonal antibodies . The table below summarizes cell line-specific detection information:

Incubation times and temperatures also affect detection sensitivity—primary antibody incubation at 4°C overnight generally yields more specific results than room temperature incubations . Enhanced chemiluminescence (ECL) detection systems provide sufficient sensitivity for most applications, though super-signal ECL may be required for detecting low abundance RUVBL1 in some cell types .

What are the key considerations for immunohistochemical detection of RUVBL1 in tissue samples?

Successful immunohistochemical detection of RUVBL1 in tissue samples depends on several critical factors that must be carefully optimized. Tissue fixation significantly impacts epitope preservation—10% neutral buffered formalin for 24-48 hours is generally recommended, as overfixation can mask RUVBL1 epitopes . Antigen retrieval is essential and should be performed using either TE buffer at pH 9.0 (preferred) or citrate buffer at pH 6.0, with heat-induced epitope retrieval at 95-98°C for 20 minutes providing optimal results . Blocking endogenous peroxidase activity with 3% hydrogen peroxide for 10 minutes before antibody application is crucial to reduce background, especially in tissues with high endogenous peroxidase activity .

Antibody dilutions should be carefully titrated for each tissue type, with a recommended starting range of 1:50-1:500 for both monoclonal and polyclonal antibodies . For optimal staining, primary antibody incubation should be performed overnight at 4°C to maximize specific binding while minimizing background signal . Detection systems should be selected based on anticipated expression levels—standard ABC (avidin-biotin complex) systems are sufficient for most applications, while polymer-based detection systems may provide enhanced sensitivity for tissues with lower RUVBL1 expression . Counterstaining with hematoxylin should be brief (1-2 minutes) to avoid obscuring RUVBL1 immunoreactivity, particularly when nuclear localization is being assessed . Tissue-specific positive controls should always be included, with mouse liver tissue and human ovarian tumor tissue having been validated for RUVBL1 detection . Negative controls should include both isotype controls and primary antibody omission to distinguish between specific staining and background .

How can researchers effectively use RUVBL1 antibodies in research related to systemic sclerosis?

Researchers investigating systemic sclerosis (SSc) can utilize RUVBL1 antibodies in several specialized applications targeting both the protein itself and autoantibodies against it. A key finding is that autoantibodies against the RuvBL1/2 complex represent a novel SSc-related autoantibody specificity found in approximately 1.1-1.9% of SSc patients across multiple cohorts . These autoantibodies are exclusively detected in SSc patients and not in disease controls or healthy subjects, making them a potential diagnostic marker .

For detecting anti-RuvBL1/2 autoantibodies in patient sera, protein immunoprecipitation assays should be employed, looking for characteristic doublets with molecular weights around 50 kDa . Liquid chromatography-mass spectrometry can be used for confirming autoantigen identity following immunoprecipitation with patient sera . When investigating the clinical associations of anti-RuvBL1/2 antibodies, researchers should note their unique association with myositis overlap and diffuse cutaneous involvement in SSc patients, as well as correlations with older age at SSc onset and male sex . Comparative studies with other SSc/myositis overlap autoantibodies (like anti-PM-Scl and anti-Ku) should be considered to distinguish the unique clinical features associated with anti-RuvBL1/2 .

For detecting RUVBL1 protein expression changes in SSc tissues, immunohistochemistry or immunofluorescence should be performed on skin biopsies from affected and unaffected areas, with standardized protocols that allow for quantitative comparison . In functional studies examining the role of RUVBL1 in SSc pathogenesis, researchers can use RUVBL1 antibodies for chromatin immunoprecipitation (ChIP) to investigate its potential involvement in transcriptional dysregulation of fibrosis-related genes .

What cross-validation techniques should be used when working with RUVBL1 antibodies?

Cross-validation is essential when working with RUVBL1 antibodies to ensure result reliability and specificity. Researchers should implement a multi-method validation approach including orthogonal detection techniques such as mass spectrometry following immunoprecipitation with RUVBL1 antibodies to confirm target identity, especially when characterizing novel interactions or modifications . Antibody comparison studies using multiple antibodies targeting different epitopes of RUVBL1 (both monoclonal and polyclonal) can help verify consistent detection patterns across experimental conditions . Genetic validation through siRNA/shRNA knockdown or CRISPR/Cas9 knockout of RUVBL1 provides critical negative controls to confirm antibody specificity, with numerous publications having already utilized RUVBL1 KD/KO systems for validation .

For functional studies, antibody-based findings should be corroborated with genetic manipulation approaches (overexpression, knockdown, or mutation) to verify that observed phenomena are truly RUVBL1-dependent . When investigating protein-protein interactions, reciprocal co-immunoprecipitation using antibodies against both RUVBL1 and its putative interaction partners provides stronger evidence for genuine interactions . For tissue expression studies, correlation of immunohistochemistry results with mRNA expression data (RT-qPCR or RNA-seq) adds an additional layer of validation . When studying post-translational modifications like ubiquitination, researchers should confirm findings using multiple approaches, such as combining immunoprecipitation-based detection with mass spectrometry analysis of the modified residues . Finally, inter-laboratory validation comparing results obtained with the same antibody across different research groups provides the highest level of confidence in antibody performance and reliability .

How can researchers address non-specific binding issues when using RUVBL1 antibodies?

Non-specific binding issues with RUVBL1 antibodies can be systematically addressed through multiple optimization strategies. If multiple bands appear in Western blots beyond the expected 50 kDa RUVBL1 band, researchers should first increase antibody dilution (starting with 2-5 fold increases) to reduce non-specific binding while maintaining specific signal . Altering blocking agents can significantly impact specificity—while 5% non-fat milk in TBST works well for most applications, switching to 3-5% BSA may reduce non-specific binding in certain sample types . Increasing wash stringency by adding 0.1-0.3% SDS to TBST wash buffer or increasing the number and duration of wash steps (minimum five washes of 5-10 minutes each) can effectively remove weakly bound antibodies .

For immunohistochemistry applications showing high background, implementing a dual blocking strategy using both protein blocking (3-5% BSA or normal serum) and chemical blocking (0.1-0.3% Triton X-100) prior to primary antibody incubation can significantly improve signal-to-noise ratio . Pre-absorption of the primary antibody with recombinant RUVBL1 protein or the immunizing peptide can help identify which bands or staining patterns represent specific binding . Using more specific secondary antibodies (highly cross-adsorbed variants) can reduce species cross-reactivity, particularly in multiplexed immunofluorescence applications . For applications with persistent non-specific binding issues, fragment antibodies (Fab or F(ab')2) derived from the original antibody may provide improved specificity by eliminating Fc-mediated interactions . Finally, validating results with alternative antibody clones targeting different epitopes of RUVBL1 can help distinguish between specific and non-specific signals, with concordant patterns across multiple antibodies suggesting true RUVBL1 detection .

What are the common pitfalls when detecting nuclear versus cytoplasmic RUVBL1?

Detecting nuclear versus cytoplasmic RUVBL1 distribution presents several common pitfalls that researchers should actively address. RUVBL1 shuttles between nuclear and cytoplasmic compartments, with distribution patterns varying by cell type, cell cycle phase, and stress conditions—particularly following irradiation or DNA damage . When performing subcellular fractionation, conventional protocols may result in cross-contamination between nuclear and cytoplasmic fractions, leading to misinterpretation of RUVBL1 localization; researchers should verify fraction purity using compartment-specific markers (e.g., lamin A/C for nucleus, GAPDH for cytoplasm) .

For immunofluorescence applications, fixation method significantly impacts apparent localization—paraformaldehyde fixation (4%, 15-20 minutes) generally preserves RUVBL1 localization better than methanol fixation, which can extract nuclear proteins . Permeabilization conditions are equally critical, with excessive detergent treatment potentially extracting loosely bound nuclear RUVBL1; optimized protocols typically use 0.1-0.3% Triton X-100 for 5-10 minutes . When performing co-localization studies, spectral bleed-through between fluorophores can create false-positive co-localization signals; single-color controls and sequential scanning are essential for accurate interpretation .

For quantitative analysis of nuclear/cytoplasmic distribution, researchers should employ digital image analysis with nuclear/cytoplasmic masks based on DAPI staining and cytoplasmic markers rather than relying on visual assessment alone . In cancer tissues, which often show aberrant nuclear/cytoplasmic distribution of RUVBL1, comparison with adjacent normal tissue using identical staining protocols is essential for accurate interpretation of localization changes . Finally, given that RUVBL1 functions in a complex with RUVBL2, parallel assessment of both proteins' localization provides more complete understanding of functional distribution patterns .

How can researchers validate RUVBL1 antibody specificity for studying the RUVBL1-DTL interaction in radioresistance?

Validating RUVBL1 antibody specificity for studying the RUVBL1-DTL interaction in radioresistance requires a multi-faceted approach focusing on both specificity and functional relevance. Antibody epitope mapping is an essential first step to ensure that the binding region is not masked during the RUVBL1-DTL interaction; epitope tags (HA, FLAG) added to recombinant RUVBL1 can help differentiate antibody detection capabilities in various binding contexts . Researchers should perform reciprocal co-immunoprecipitation using both RUVBL1 and DTL antibodies to confirm the interaction, with consistent detection in both directions providing stronger evidence for genuine interaction .

Competition assays using purified recombinant RUVBL1 protein to block antibody binding in immunoprecipitation or immunofluorescence experiments can help verify antibody specificity in the context of DTL interaction studies . For radiation response studies, researchers should validate that the selected RUVBL1 antibody effectively detects both non-modified and K63-ubiquitinated forms of RUVBL1, as the latter is critical for the DTL-mediated radioresistance mechanism . Western blot analysis comparing RUVBL1 detection before and after DTL knockdown/overexpression can confirm the antibody's ability to detect DTL-dependent changes in RUVBL1 modification status .

Proximity ligation assays (PLA) using antibodies against both RUVBL1 and DTL provide spatial validation of the interaction in situ, with quantification of PLA signals before and after radiation treatment to assess radiation-induced interaction dynamics . Mass spectrometry validation following RUVBL1 immunoprecipitation can comprehensively identify DTL-dependent post-translational modifications and interaction sites, providing gold-standard confirmation of antibody-based findings . Finally, functional validation through genetic manipulation (RUVBL1 or DTL knockdown/knockout) combined with radiation sensitivity assays can confirm the biological relevance of antibody-detected interactions and modifications in radioresistance mechanisms .

How can researchers integrate findings across multiple experimental platforms using RUVBL1 antibodies?

Integrating findings across multiple experimental platforms requires systematic correlation of RUVBL1 antibody-derived data with complementary methodologies. Researchers should establish cross-platform validation protocols that correlate protein expression data from Western blotting with transcriptomic analyses (RT-qPCR or RNA-seq) to differentiate between transcriptional and post-transcriptional regulation mechanisms affecting RUVBL1 levels . Integration of immunofluorescence localization data with biochemical fractionation results provides more robust evidence of subcellular distribution patterns, particularly important when studying nuclear-cytoplasmic shuttling during DNA damage response .

For interaction studies, researchers should correlate co-immunoprecipitation findings with proximity-based methods like BioID, APEX, or proximity ligation assays to build comprehensive protein interaction networks around RUVBL1 . When studying chromatin association and transcriptional regulation, integration of ChIP-seq data (using RUVBL1 antibodies) with expression profiling of target genes creates more complete mechanistic understanding of RUVBL1's regulatory functions . For clinical samples, correlation of immunohistochemical findings with patient outcomes and molecular subtypes (determined by genomic/transcriptomic profiling) can reveal clinically relevant associations, as demonstrated in the SSc studies identifying anti-RUVBL1/2 autoantibodies as markers for specific disease subsets .