RUVBL1 Antibody

What is RUVBL1 and why is it important in biological research?

RUVBL1 is a 49-50 kDa nuclear protein that functions as part of various multiprotein complexes involved in DNA repair and transcriptional regulation. It has structural similarities to bacterial RuvB, which is a DNA helicase . In research settings, RUVBL1 has gained importance due to:

• Its essential role in multiple cellular processes including chromatin remodeling and DNA damage response

• Its involvement in the INO80 complex and other chromatin-modifying complexes

• Its emerging role in autoimmune conditions when it becomes a target of autoantibodies

• Its potential manipulation by viruses, particularly adenovirus, to suppress immune responses

RUVBL1 is ubiquitously expressed, with highest expression observed in heart, skeletal muscle, and testis tissues . The protein's involvement in numerous cellular pathways makes it a significant target for studies investigating fundamental cellular processes and disease mechanisms.

What detection methods are most reliable for RUVBL1 in different sample types?

For detecting RUVBL1 in research samples, several methodological approaches can be employed:

Western Blot (WB): The most common method for detecting RUVBL1 protein expression levels. When using antibodies like DF7961, researchers should expect a band at approximately 50 kDa, with a possible isoform at 43 kDa . Optimization steps include:

• Using appropriate lysis buffers containing protease inhibitors

• Loading 20-40 μg of total protein

• Transferring at lower voltage for longer periods due to the protein's size

• Blocking with 5% non-fat milk or BSA

Immunohistochemistry (IHC): Effective for localization studies in tissue samples. Both paraffin-embedded and frozen sections can be used .

Immunofluorescence/Immunocytochemistry (IF/ICC): Ideal for subcellular localization studies, typically showing a predominantly nuclear speckled pattern with some cytoplasmic staining in approximately 40% of cells .

Particle Multi Analyte Technology (PMAT): A specialized technique that allows simultaneous detection of multiple autoantibodies, including anti-RUVBL1/2. This approach is particularly valuable for clinical research involving autoimmune conditions .

What controls should be included when performing experiments with RUVBL1 antibodies?

To ensure experimental validity when working with RUVBL1 antibodies, the following controls are recommended:

Positive Controls:

• Heart, skeletal muscle, or testis tissue lysates, which express high levels of RUVBL1

• Cell lines known to express RUVBL1 (most human cell lines express detectable levels)

• Recombinant RUVBL1 protein for antibody validation

Negative Controls:

• RUVBL1 knockout/knockdown samples

• Secondary antibody-only controls to assess non-specific binding

• Pre-immune serum (for polyclonal antibodies)

• Isotype controls (for monoclonal antibodies)

Specificity Controls:

• Peptide competition assays to confirm antibody specificity

• Cross-reactivity testing against the related RUVBL2 protein, which shares structural similarities

When examining autoantibodies against RUVBL1/2, as in clinical research, include control samples from healthy donors and disease controls to establish appropriate cutoff values and confirm specificity (99.2% specificity has been reported for PMAT-based detection) .

How can researchers differentiate between anti-RUVBL1 and anti-RUVBL2 autoantibodies in patient samples?

Differentiating between anti-RUVBL1 and anti-RUVBL2 autoantibodies requires specialized techniques:

Particle Multi Analyte Technology (PMAT):
This advanced technique uses paramagnetic particles coupled with recombinant full-length RUVBL1 and RUVBL2 proteins that carry unique signatures. The process involves:

• Incubating diluted serum samples with these particles

• Washing and adding PE-conjugated anti-human IgG

• Measuring median fluorescence intensity (MFI)

• Analyzing results using proprietary algorithms to distinguish between the two autoantibodies

When establishing cutoffs, researchers should test against disease control samples to ensure specificity. In published studies, a 99.2% specificity was obtained using preliminary cutoffs for RUVBL1 and RUVBL2 assays .

Immunoprecipitation:
The original method for identifying these autoantibodies involves:

• Incubating patient sera with cell lysates

• Precipitating immune complexes

• Separating by SDS-PAGE

• Identifying distinct bands at approximately 50 kDa (49 kD for RUVBL1 and 48 kD for RUVBL2)

Western Blot with Recombinant Proteins:
Using purified recombinant RUVBL1 and RUVBL2 proteins in parallel Western blots can help distinguish antibodies against each protein based on their slightly different migration patterns.

What is the clinical significance of anti-RUVBL1/2 autoantibodies in systemic sclerosis and overlapping syndromes?

Anti-RUVBL1/2 autoantibodies have emerged as important biomarkers in systemic sclerosis (SSc) and related conditions. Based on current literature analyzing 52 reported cases, the following clinical associations have been observed:

Demographic and Disease Associations:

• More frequently found in older patients of male sex

• Highly specific for SSc, particularly associated with SSc/polymyositis (PM) overlaps and diffuse SSc

• Approximately 60% of cases present with overlap syndromes (mainly "scleromyositis")

• About 35% present with SSc alone

• Rare cases include SSc/Sjögren's overlap and morphea/myositis overlap

Organ Involvement (% of patients):

Laboratory Findings:

• Speckled ANA pattern (AC-4) on HEp-2 cells with characteristic variation during cell cycle phases (increased in prophase, decreased in metaphase)

• Fine-speckled cytoplasmic pattern in approximately 40% of positive sera

• In 92% of cases, anti-RUVBL1/2 autoantibodies are isolated, with few cases overlapping with other SSc/myositis autoantibodies (RNAP3, Ku, Th/T0)

This serological profile helps identify a distinct subset of SSc patients with particular clinical features, allowing for more tailored monitoring and management approaches.

How does RUVBL1 contribute to viral immune evasion strategies, particularly in adenovirus infection?

RUVBL1 plays a significant role in viral immune evasion, particularly regarding type I interferon signaling during adenovirus infection:

Mechanism of Adenovirus E1A-RUVBL1 Interaction:

• The adenovirus immediate-early gene E1A binds to RUVBL1 via its C-terminal domain (residues 224-254)

• This interaction recruits E1A to RUVBL1-regulated interferon-stimulated gene (ISG) promoters

• E1A prevents activation of these promoters, thereby suppressing the cellular interferon response

Functional Consequences:

• Depletion of RUVBL1 reduces adenovirus growth by more than 3-fold

• This growth reduction occurs despite no apparent direct effect on viral gene expression at early infection stages (24h and earlier)

• Viral gene expression is reduced at later timepoints (48-72h) in RUVBL1-depleted cells

• RUVBL1 is not directly recruited to viral promoters, suggesting its role is primarily in modulating host interferon responses rather than viral transcription

Research Implications:
RUVBL1 represents a novel target for adenovirus in its strategy to suppress interferon responses. This adds to our understanding of viral immune evasion mechanisms and identifies a potential target for antiviral interventions. Researchers examining viral host interactions should consider RUVBL1's role when studying immune evasion strategies of DNA viruses.

What are the optimal experimental conditions for detecting RUVBL1 using antibodies in Western blot applications?

For optimal Western blot detection of RUVBL1, researchers should consider the following protocol parameters:

Sample Preparation:

• Lyse cells in RIPA buffer supplemented with protease inhibitors

• Include phosphatase inhibitors if interested in phosphorylation status

• Sonicate briefly to shear DNA and reduce sample viscosity

• Heat samples at 95°C for 5 minutes in Laemmli buffer containing DTT or β-mercaptoethanol

Gel Electrophoresis:

• Use 10-12% SDS-PAGE gels for optimal resolution around 50 kDa

• Load 20-40 μg of total protein per lane

• Include molecular weight markers spanning 25-75 kDa range

• Run at 100-120V to ensure good band resolution

Transfer Conditions:

• Use PVDF membrane (0.45 μm pore size) for better protein retention

• Transfer at lower voltage (30V) overnight at 4°C for efficient transfer of the 50 kDa protein

• Confirm transfer efficiency with reversible protein stains (Ponceau S)

Antibody Incubation:

• Block membrane with 5% non-fat milk in TBST for 1 hour at room temperature

• Dilute primary antibody (like DF7961) according to manufacturer recommendations, typically 1:1000-1:2000

• Incubate overnight at 4°C with gentle agitation

• Wash 4-5 times with TBST, 5 minutes each

• Incubate with appropriate HRP-conjugated secondary antibody (1:5000-1:10000) for 1 hour at room temperature

• Perform final washes (4-5 times) with TBST

Detection:

• Use enhanced chemiluminescence (ECL) substrate appropriate for the expected signal intensity

• Expose to film or use digital imaging systems with exposure times optimized for the 50 kDa band

• Expect bands at approximately 50 kDa, with potential isoforms at 43 kDa

Validation Controls:

• Include positive control (heart or testis tissue lysate)

• Consider including RUVBL1 knockdown/knockout controls

• For non-human samples, ensure cross-reactivity with the antibody used (confirmed for mouse and monkey samples with DF7961)

How can researchers accurately distinguish between RUVBL1 and RUVBL2 proteins in experimental systems?

Antibody-Based Approaches:

• Use highly specific monoclonal antibodies raised against unique epitopes

• Validate antibody specificity using recombinant proteins and knockout/knockdown controls

• Perform peptide competition assays to confirm specificity

• Consider dual-color Western blotting with differentially labeled antibodies against each protein

Mass Spectrometry:

• Use targeted proteomic approaches to identify unique peptides for each protein

• Develop selected reaction monitoring (SRM) or parallel reaction monitoring (PRM) assays for quantitative discrimination

• Analyze post-translational modifications which may differ between the two proteins

Genetic Manipulation:

• Use siRNA or CRISPR/Cas9 to selectively knock down each protein

• Create tagged versions (with different tags) for overexpression studies

• Use inducible systems to control the expression of each protein independently

Functional Assays:

• Design assays targeting known functional differences

• Assess ATPase activity under different conditions

• Evaluate interactions with proteins known to preferentially bind one over the other

Expression Analysis:

• Compare expression patterns across tissues

• Examine relative expression levels in pathological states, particularly autoimmune conditions where they serve as autoantigens

When analyzing clinical samples for autoantibodies, using the PMAT technology with separate RUVBL1 and RUVBL2 antigens coupled to differently labeled paramagnetic particles allows for simultaneous but distinct detection of antibodies against each protein .

What is the importance of anti-RUVBL1/2 testing in patients with suspected overlap syndromes?

Testing for anti-RUVBL1/2 autoantibodies has significant clinical research value in patients with suspected overlap syndromes, particularly those with features of both systemic sclerosis (SSc) and inflammatory myopathies:

Diagnostic Value:

• Anti-RUVBL1/2 antibodies are highly specific for SSc and SSc/myositis overlap syndromes

• They can be detected in cases where conventional antibody testing is negative

• They exhibit a distinctive speckled pattern on HEp-2 cells that can serve as a screening indicator

• Detection may facilitate earlier diagnosis of overlap conditions

Clinical Phenotype Prediction:
Based on studies of 52 reported cases, patients with anti-RUVBL1/2 autoantibodies demonstrate a characteristic pattern of organ involvement:

• Very high rates of gastrointestinal involvement (94%)

• High prevalence of interstitial lung disease (88%)

• Significant cardiac involvement in approximately half of patients

• Myopathy, particularly in overlap cases

• Notably low rates of pulmonary arterial hypertension and severe renal involvement

Testing Methodology:
For clinical research applications, the preferred testing approach is:

• Initial screening with indirect immunofluorescence on HEp-2 cells (look for characteristic fine-speckled pattern with cell cycle variation)

• Confirmation using specialized techniques such as:

  • Particle multi analyte technology (PMAT)

  • Immunoprecipitation

  • Line immunoassays with purified antigens

These testing modalities have demonstrated high specificity (99.2% in one study) when appropriate cutoffs are established using disease control samples .

How can researchers integrate RUVBL1 antibody testing into multi-analyte autoantibody profiling platforms?

The integration of RUVBL1 antibody testing into multi-analyte platforms represents an advanced approach for comprehensive autoantibody profiling in research settings:

Particle Multi Analyte Technology (PMAT) Implementation:
This innovative technology allows for simultaneous detection of multiple autoantibodies, including anti-RUVBL1 and anti-RUVBL2:

• Assay Design:

  • Couple recombinant full-length RUVBL1 protein to paramagnetic particles with unique signatures

  • Include multiple other autoantigens relevant to connective tissue diseases on different particles

  • The reported CTD research panel includes 32+ antigens (dsDNA, RNP, Sm, Ro60, Ro52, SS-B, centromere, Scl-70, Jo-1, etc.)

• Protocol Overview:

  • Incubate diluted serum samples with the paramagnetic particles (9.5 minutes at 37°C)

  • Wash and incubate with PE-conjugated anti-human IgG (9.5 minutes at 37°C)

  • Analyze median fluorescence intensity (MFI) using digital imaging and proprietary algorithms

• Validation Requirements:

  • Establish preliminary cutoffs based on disease control screening

  • Verify specificity using diverse control populations (reported 99.2% specificity)

  • Incorporate internal calibrators and controls for standardization across runs

Integration with Other Platforms:

• Consider combining PMAT with other methodologies (immunoprecipitation, line blots) for confirmatory analysis

• Develop algorithms for interpretation of complex autoantibody profiles

• Correlate results with clinical data to establish clinically relevant cutoffs

Research Applications:

• Cohort studies of autoimmune diseases

• Longitudinal monitoring of autoantibody profiles

• Correlation of autoantibody patterns with clinical manifestations

• Biomarker discovery in novel autoimmune phenotypes

Multi-analyte approaches significantly enhance research efficiency by enabling simultaneous detection of numerous autoantibodies from limited sample volumes, providing comprehensive autoantibody profiling while conserving precious research specimens.

What are common troubleshooting approaches for inconsistent RUVBL1 antibody signals in Western blotting?

Researchers working with RUVBL1 antibodies may encounter signal inconsistencies. Here are methodological solutions to common problems:

Problem: Weak or Absent Signal

• Solution 1: Optimize protein extraction using nuclear extraction protocols, as RUVBL1 is primarily nuclear. Include appropriate detergents (0.5-1% NP-40 or Triton X-100) in lysis buffers.

• Solution 2: Increase antibody concentration or extend incubation time (overnight at 4°C).

• Solution 3: Enhance detection sensitivity using high-sensitivity ECL substrates or signal amplification systems.

• Solution 4: Verify sample integrity by probing for housekeeping proteins.

• Solution 5: Check antibody compatibility with your sample species (DF7961 is confirmed for human, mouse, and monkey samples) .

Problem: Multiple Bands or High Background

• Solution 1: Increase blocking stringency (5-10% milk or BSA) and extend blocking time.

• Solution 2: Add 0.1-0.3% Tween-20 in washing buffers and increase wash frequency/duration.

• Solution 3: Dilute primary antibody further and use fresher antibody aliquots.

• Solution 4: Pre-adsorb antibody with cell lysate from non-expressing cells.

• Solution 5: Consider that the 43 kDa band may represent a legitimate isoform of RUVBL1 .

Problem: Inconsistent Results Between Experiments

• Solution 1: Standardize protein quantification methods and loading amounts.

• Solution 2: Prepare master mixes of antibody dilutions to ensure consistency.

• Solution 3: Control for post-translational modifications by adding appropriate inhibitors to lysis buffers.

• Solution 4: Standardize exposure times and image acquisition settings.

• Solution 5: Include positive control samples in every experiment.

Problem: Differential Detection Between Cell Types

• Solution 1: Adjust lysis conditions for different cell types (adherent vs. suspension).

• Solution 2: Consider nuclear extraction for cells with lower expression levels.

• Solution 3: Verify expression levels using RT-qPCR as a complementary approach.

• Solution 4: Account for tissue-specific expression patterns (higher in heart, skeletal muscle, and testis) .

How can researchers study the functional interaction between RUVBL1 and type I interferon signaling pathways?

Investigating the functional interaction between RUVBL1 and type I interferon signaling requires sophisticated experimental approaches:

Genetic Manipulation Strategies:

• CRISPR/Cas9 Knockout: Generate RUVBL1-deficient cell lines to assess impact on interferon-stimulated gene (ISG) expression.

• siRNA/shRNA Knockdown: Use RNA interference for transient or stable RUVBL1 depletion, as demonstrated in studies showing >3-fold reduction in adenovirus growth after RUVBL1 depletion .

• Overexpression Systems: Create cell lines with wild-type or mutant RUVBL1 to assess functional domains important for interferon signaling.

• Domain Mapping: Generate truncation or point mutants to identify critical regions for interaction with interferon pathway components.

Protein-Protein Interaction Studies:

• Co-immunoprecipitation: Assess interaction between RUVBL1 and components of interferon signaling pathway.

• Proximity Ligation Assay: Visualize and quantify endogenous protein interactions in situ.

• FRET/BRET Approaches: Measure real-time dynamics of protein associations.

• Mass Spectrometry: Identify RUVBL1 interactome changes following interferon stimulation.

Chromatin Association Analysis:

• Chromatin Immunoprecipitation (ChIP): Assess RUVBL1 recruitment to interferon-stimulated gene promoters, as was attempted in viral studies .

• ChIP-seq: Genome-wide mapping of RUVBL1 binding sites before and after interferon stimulation.

• CUT&RUN/CUT&Tag: Higher resolution alternatives to ChIP for mapping protein-DNA interactions.

• ATAC-seq: Assess chromatin accessibility changes dependent on RUVBL1 at interferon-regulated loci.

Functional Readouts:

• Luciferase Reporter Assays: Measure interferon-stimulated response element (ISRE) activity in presence/absence of RUVBL1.

• RT-qPCR Panels: Quantify expression changes in key ISGs following RUVBL1 manipulation.

• Viral Replication Assays: Measure growth of interferon-sensitive viruses as a functional readout.

• RNA-seq: Assess global transcriptional changes in interferon response upon RUVBL1 modulation.

Viral Interference Models:

• Adenovirus E1A Studies: Investigate how E1A binding to RUVBL1 (via residues 224-254) affects interferon signaling .

• Mutant Virus Studies: Compare wild-type adenovirus to E1A mutants deficient in RUVBL1 binding.

• Host-Pathogen Interaction Screens: Identify other viral factors that may target RUVBL1.

These methodological approaches provide a comprehensive toolkit for dissecting RUVBL1's role in type I interferon signaling, with particular relevance to viral immune evasion strategies.

What emerging applications of RUVBL1 antibodies show promise in autoimmune disease research?

The study of RUVBL1 and its antibodies in autoimmune conditions is an evolving field with several promising research directions:

Biomarker Development:

• Integration of anti-RUVBL1/2 antibody testing into comprehensive autoantibody panels for early detection of overlap syndromes

• Development of standardized, commercially available test kits with validated cutoffs

• Longitudinal studies to determine if antibody titers correlate with disease activity or predict organ involvement

• Investigation of anti-RUVBL1/2 in seronegative patients with clinical features suggestive of connective tissue diseases

Mechanistic Studies:

• Exploration of why RUVBL1/2 becomes an autoantigen in specific patient subsets

• Investigation of potential trigger events leading to loss of immune tolerance to these nuclear proteins

• Assessment of whether post-translational modifications of RUVBL1/2 contribute to autoantigenicity

• Study of epitope spreading in the development of anti-RUVBL1/2 responses

Therapeutic Targeting:

• Development of inhibitors or modulators of RUVBL1 function for potential therapeutic applications

• Investigation of whether targeting RUVBL1-mediated pathways could modify autoimmune disease progression

• Exploration of the intersection between RUVBL1's role in interferon signaling and autoimmunity

• Assessment of RUVBL1 as a potential therapeutic target in interferon-driven autoimmune diseases

Expanded Clinical Phenotyping:

• Larger cohort studies to better define the clinical spectrum associated with anti-RUVBL1/2 antibodies

• Investigation of potential ethnic or geographical variations in antibody prevalence and associated phenotypes

• More detailed assessment of treatment responses in anti-RUVBL1/2-positive patients

• Study of potential associations with malignancy, as observed with other myositis-specific antibodies

These emerging research directions highlight the potential of RUVBL1 antibodies as tools for advancing our understanding of autoimmune disease mechanisms and improving patient stratification for clinical research.

How might advances in multiplexed imaging techniques enhance RUVBL1 localization studies?

Recent advances in multiplexed imaging technologies offer unprecedented opportunities for studying RUVBL1 localization and interactions at subcellular resolution:

Multiplex Immunofluorescence Approaches:

• Cyclic Immunofluorescence (CycIF): This technique allows for sequential staining, imaging, and signal removal, enabling visualization of 30+ proteins within the same sample. For RUVBL1 studies, this could reveal co-localization with different chromatin-modifying complexes or interferon signaling components in various cellular states.

• CO-Detection by indEXing (CODEX): Using DNA-barcoded antibodies, CODEX enables highly multiplexed imaging of 40+ targets simultaneously, allowing researchers to map RUVBL1's dynamic interactions with multiple proteins during different cellular processes.

• Imaging Mass Cytometry (IMC): By combining mass spectrometry with imaging, IMC can measure 40+ proteins simultaneously in tissue sections with subcellular resolution, enabling detailed mapping of RUVBL1 distribution across tissue microenvironments.

Super-Resolution Microscopy Applications:

• Stochastic Optical Reconstruction Microscopy (STORM): With ~20 nm resolution, STORM can resolve RUVBL1 distribution within nuclear subcompartments and potentially distinguish between different RUVBL1-containing complexes.

• Stimulated Emission Depletion (STED) Microscopy: Offering resolution down to 30-50 nm, STED could reveal precise spatial relationships between RUVBL1 and its interaction partners during processes like DNA damage repair.

• Expansion Microscopy: By physically expanding specimens while maintaining relative protein positions, this technique allows visualization of molecular complexes with conventional microscopes, potentially revealing novel aspects of RUVBL1 organization.

Live-Cell Imaging Innovations:

• Lattice Light-Sheet Microscopy: Enables gentle, high-speed 3D imaging of living cells, ideal for tracking RUVBL1 dynamics during cellular responses to interferon or viral infection.

• CRISPR-based Fluorescent Tagging: Endogenous tagging of RUVBL1 with fluorescent proteins to track native expression and localization without overexpression artifacts.

• Optogenetic Approaches: Light-inducible protein interactions could be used to manipulate RUVBL1 associations in real-time while monitoring cellular responses.

Correlative Light and Electron Microscopy (CLEM):

• Combining fluorescence microscopy with electron microscopy allows researchers to locate RUVBL1 using antibody-based fluorescence imaging and then examine the ultrastructural context at nanometer resolution.

These advanced imaging approaches could significantly enhance our understanding of RUVBL1's dynamic localization and interactions in both normal cellular processes and disease states, particularly in contexts like interferon signaling and viral infection where RUVBL1 plays important regulatory roles.