RUVBL2 Antibody, HRP conjugated

What is RUVBL2 and why is it important in cellular function?

RUVBL2 is a member of the AAA+ (ATPases Associated with diverse cellular Activities) family of proteins with a molecular weight of approximately 51 kDa. It serves as a critical component in numerous cellular processes including DNA damage repair, chromatin remodeling, and transcriptional regulation. RUVBL2 often functions in complex with RUVBL1, forming the RUVBL1/2 complex that participates in various multi-protein assemblies. This protein's importance lies in its diverse roles in cellular homeostasis, with its dysregulation implicated in various pathological conditions including cancer and neurodegenerative disorders . RUVBL2 forms part of the PAQosome/R2TP complex responsible for RNA polymerase II assembly in the cytoplasm, demonstrating its critical role in transcriptional machinery formation .

What types of RUVBL2 antibodies are commercially available for research?

Research-grade RUVBL2 antibodies are available in several formats to accommodate diverse experimental needs. Primary antibodies include polyclonal antibodies such as the Reptin/RUVBL2 Rabbit Polyclonal Antibody (CAB12564) and the RUVBL2 antibody (10195-1-AP) . These are typically unconjugated primary antibodies that require secondary detection methods. HRP-conjugated versions combine the specificity of anti-RUVBL2 antibodies with the enzymatic activity of horseradish peroxidase for direct detection in applications such as Western blotting, eliminating the need for secondary antibodies. The most commonly available RUVBL2 antibodies have been generated in rabbits and demonstrate reactivity with human, mouse, and rat samples .

What are the primary applications for RUVBL2 antibodies in research?

RUVBL2 antibodies serve as versatile tools for investigating this protein's expression, localization, and interactions across various experimental contexts. Common applications include:

• Western Blot (WB): Detection of RUVBL2 protein expression levels in cell or tissue lysates (recommended dilution 1:500-1:2000)

• Immunohistochemistry (IHC): Visualization of RUVBL2 distribution in tissue sections (recommended dilution 1:250-1:1000)

• Immunofluorescence/Immunocytochemistry (IF/ICC): Subcellular localization studies (recommended dilution 1:200-1:800)

• Immunoprecipitation (IP): Isolation of RUVBL2 and associated protein complexes

• ELISA: Quantitative detection of RUVBL2 in solution

HRP-conjugated versions are particularly advantageous for Western blotting applications, offering enhanced sensitivity and simplified workflows by eliminating secondary antibody steps.

How can researchers effectively validate the specificity of RUVBL2 antibodies in their experimental system?

Validating antibody specificity is crucial for generating reliable research data. For RUVBL2 antibodies, including HRP-conjugated versions, a comprehensive validation approach should include:

• Positive and negative controls: Use cell lines with known RUVBL2 expression profiles. Positive controls documented in literature include C2C12, Daudi, HeLa, and HepG2 cells .

• Knockdown/knockout validation: Compare antibody signal between wild-type samples and those with reduced RUVBL2 expression (siRNA/shRNA knockdown) or CRISPR-Cas9-mediated knockout. This represents the gold standard for specificity verification.

• Molecular weight confirmation: RUVBL2 should appear at approximately 51 kDa on Western blots. Deviation from this expected position requires further investigation .

• Multiple antibody comparison: Use antibodies targeting different epitopes of RUVBL2 to confirm consistent detection patterns.

• Cross-reactivity assessment: Test the antibody against recombinant RUVBL1 to ensure it doesn't cross-react with this closely related protein, particularly important since these proteins often function together .

• Peptide competition assay: Pre-incubate the antibody with the immunogen peptide to confirm signal abolishment in subsequent detection assays.

What are the considerations for optimizing immunoblot detection of RUVBL2 using HRP-conjugated antibodies?

Optimizing immunoblot detection of RUVBL2 with HRP-conjugated antibodies requires attention to several parameters:

• Sample preparation: Complete cell lysis is essential given RUVBL2's involvement in multi-protein complexes. Consider using buffer systems containing non-ionic detergents (0.5-1% NP-40 or Triton X-100) supplemented with protease inhibitors.

• Loading control selection: GAPDH serves as a reliable loading control for RUVBL2 detection, as demonstrated in multiple studies .

• Blocking optimization: Use 5% non-fat dry milk or BSA in TBST. For HRP-conjugated antibodies, BSA may be preferable to avoid biotin-containing proteins in milk that can increase background.

• Antibody dilution: Although general recommendations exist (1:500-1:2000), optimal dilution for HRP-conjugated versions may differ and should be empirically determined for each experimental system .

• Incubation conditions: For primary HRP-conjugated antibodies, overnight incubation at 4°C typically yields the best signal-to-noise ratio, but shorter incubations (1-2 hours) at room temperature may be sufficient with concentrated antibody stocks.

• Detection system selection: Enhanced chemiluminescence (ECL) systems vary in sensitivity. Standard ECL is sufficient for abundant targets, while femto-level systems may be necessary for low-abundance detection.

• Membrane washing: Thorough washing (4-5 times with TBST for 5-10 minutes each) is critical to minimize background with direct HRP detection systems.

How do post-translational modifications of RUVBL2 impact antibody recognition and experimental design?

Post-translational modifications (PTMs) of RUVBL2 can significantly affect antibody recognition, necessitating careful consideration in experimental design:

• Phosphorylation: RUVBL2 undergoes phosphorylation at multiple sites, which can alter its conformation and potentially mask antibody epitopes. Phosphorylation status varies depending on cell cycle stage and cellular stress conditions.

• SUMOylation: This modification can affect RUVBL2's nuclear localization and complex formation capabilities.

• Epitope accessibility: When RUVBL2 functions within multi-protein complexes like PAQosome/R2TP, certain epitopes may become inaccessible to antibodies .

• Antibody selection strategy:

  • For total RUVBL2 detection regardless of modification status, select antibodies targeting regions less likely to undergo PTMs

  • For PTM-specific detection, use modification-specific antibodies in conjunction with total RUVBL2 antibodies

  • Consider using multiple antibodies recognizing different epitopes to obtain a complete picture of RUVBL2 expression and modification status

• Sample preparation: Different lysis conditions may preserve or disrupt certain PTMs. Phosphatase inhibitors should be included when studying phosphorylation states.

What protocols are recommended for studying RUVBL2 interactions with transcriptional machinery?

For investigating RUVBL2's role in transcriptional regulation, particularly its reported function in RNA polymerase II (Pol II) clustering and assembly , the following methodological approaches are recommended:

• Co-immunoprecipitation (Co-IP) protocol:

  • Crosslink cells with 1% formaldehyde for 10 minutes to preserve transient interactions

  • Lyse cells in buffer containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, with protease and phosphatase inhibitors

  • Pre-clear lysate with protein A/G beads

  • Incubate with anti-RUVBL2 antibody overnight at 4°C

  • Add protein A/G beads, wash extensively, and elute

  • Analyze by immunoblotting with antibodies against suspected interaction partners (e.g., Pol II subunits)

• Chromatin Immunoprecipitation (ChIP) approach:

  • This method is particularly valuable for identifying genomic binding sites of RUVBL2 in association with transcription factors

  • Focus on known target genes such as c-Myc, Bmp4, and Junb

  • Compare results using both anti-RUVBL2 antibodies and antibodies against specific transcription factors

• Proximity Ligation Assay (PLA):

  • Optimal for visualizing in situ protein-protein interactions

  • Use anti-RUVBL2 antibody in combination with antibodies against transcription factors or Pol II subunits

  • Secondary antibodies conjugated with complementary oligonucleotides generate fluorescent signals when proteins are in close proximity (<40 nm)

  • Particularly useful for studying dynamic interactions during transcriptional activation

How should researchers approach experimental design when studying RUVBL2 in disease models, particularly autoimmune conditions?

When investigating RUVBL2 in disease contexts, especially autoimmune conditions where RUVBL1/2 autoantibodies have been reported , researchers should consider the following methodological approaches:

• Patient sample analysis protocol:

  • For autoantibody detection in systemic sclerosis (SSc):

    • Use protein immunoprecipitation assays with radiolabeled cell extracts

    • Confirm with immunoblotting using purified recombinant RUVBL1/2

    • Consider liquid chromatography-mass spectrometry for precise autoantigen characterization

• Experimental cohort design:

  • Include appropriate disease controls (e.g., SLE, PM, DM, RA) alongside healthy controls

  • For SSc studies, follow the approach used in published cohorts (Kanazawa, Keio, and Pittsburgh)

  • Ensure sufficient statistical power based on reported antibody prevalence (~1.1-1.9% in SSc patients)

• Correlation with clinical parameters:

  • Document comprehensive clinical data including disease subset, organ involvement, and laboratory parameters

  • Analyze using appropriate statistical methods for rare autoantibody specificities

  • Consider longitudinal sampling to assess temporal relationships with disease progression

• Functional studies in relevant cellular models:

  • Establish whether patient-derived autoantibodies affect RUVBL2 function

  • Consider in vitro transcription assays to assess impact on RUVBL2's role in transcriptional regulation

  • Evaluate effects on Pol II clustering and assembly in cellular models

What techniques are recommended for studying RUVBL2's role in phase separation and transcriptional condensate formation?

Recent research has identified RUVBL2 as a regulator of phase separation and transcriptional condensate formation . The following methodological approaches are recommended for investigating this emerging aspect of RUVBL2 biology:

• In vitro phase separation assays:

  • Express and purify recombinant RUVBL2 and potential interaction partners (e.g., RNA Pol II CTD)

  • Assess phase separation under varying conditions (protein concentration, salt concentration, temperature)

  • Use fluorescently labeled proteins to visualize droplet formation by fluorescence microscopy

  • Quantify droplet size, number, and fusion events

• Cellular condensate visualization:

  • Use fluorescently tagged RUVBL2 (ensure tag doesn't interfere with function)

  • Employ live-cell imaging to monitor dynamics of condensate formation

  • Consider FRAP (Fluorescence Recovery After Photobleaching) to assess protein mobility within condensates

  • Correlate with transcriptional activity using nascent RNA labeling techniques

• Perturbation approaches:

  • RUVBL2 overexpression or depletion to assess effects on Pol II clustering

  • Targeted mutagenesis of domains involved in phase separation

  • Chemical disruption of condensates (e.g., 1,6-hexanediol treatment)

  • Assessment of transcriptional outcomes using nascent RNA sequencing

• Time-resolved analysis:

  • Implement time series analysis following rapid protein degradation systems

  • Monitor both nascent and mature transcriptomes to distinguish direct from indirect effects

  • Focus on established RUVBL2-dependent genes (c-Myc, Bmp4, Junb)

What are common sources of variability in RUVBL2 detection and how can they be mitigated?

Researchers may encounter several sources of variability when detecting RUVBL2 with antibodies, including HRP-conjugated versions. Understanding and addressing these factors is crucial for reliable data interpretation:

• Complex-dependent epitope masking:

  • RUVBL2 functions within various protein complexes (e.g., PAQosome/R2TP)

  • This can lead to inconsistent detection depending on complex integrity in sample preparation

  • Solution: Compare multiple lysis conditions with varying detergent stringency to optimize epitope accessibility

• Expression level variations:

  • RUVBL2 expression may vary across cell types and disease states

  • In C9orf72-ALS/FTD patient-derived cells, altered RUVBL levels have been reported

  • Solution: Include appropriate positive controls with known RUVBL2 expression levels (e.g., HeLa, HepG2 cells)

• Antibody lot-to-lot variability:

  • Particularly relevant for polyclonal antibodies, including HRP-conjugated versions

  • Solution: Record lot numbers and validate each new lot against previous standards

• Cross-reactivity concerns:

  • RUVBL1 and RUVBL2 share structural similarities that may lead to cross-reactivity

  • Solution: Include controls expressing only RUVBL1 or RUVBL2 to confirm specificity

• HRP conjugation effects:

  • Direct HRP conjugation may affect antibody binding properties or sensitivity

  • Solution: When transitioning from unconjugated to HRP-conjugated antibodies, re-optimize working dilutions and incubation conditions

• Storage and handling considerations:

  • HRP activity can diminish over time or with improper storage

  • Solution: Aliquot antibodies to minimize freeze-thaw cycles and store at recommended temperatures (typically -20°C with glycerol)

How should researchers interpret contradictory results between different detection methods when studying RUVBL2?

When faced with discrepancies in RUVBL2 detection across different methodologies, consider the following analytical framework:

• Method-specific biases:

  • Western blot: Detects denatured protein, potentially missing conformation-dependent features

  • Immunofluorescence: Preserves cellular context but may have fixation-dependent artifacts

  • Immunoprecipitation: Maintains protein complexes but may miss transient interactions

  Solution: View each method as providing complementary rather than redundant information

• Systematic analysis approach:

  Method	Strengths	Limitations	Best Application
  Western Blot (HRP)	Quantitative, specific to molecular weight	Loses spatial information	Protein level quantification
  Immunofluorescence	Preserves spatial context	Generally qualitative	Localization studies
  Immunoprecipitation	Captures interaction partners	Potential for non-specific binding	Complex composition analysis
  ChIP	Identifies genomic binding sites	Indirect DNA association	Transcriptional regulation studies

• Resolution strategies:

  • For discrepancies between protein levels (WB) and localization (IF): Consider fraction-specific analysis

  • For contradictions between expression and activity: Implement functional assays

  • For inconsistencies across antibodies: Target different epitopes to rule out PTM-dependent effects

• Triangulation with orthogonal methods:

  • Supplement antibody-based detection with mass spectrometry

  • Validate with genetic approaches (knockdown/overexpression)

  • Consider mRNA expression correlation analysis

How can RUVBL2 antibodies be leveraged to study its role in neurodegenerative diseases?

Recent research has implicated RUVBL1/2 in reducing toxic dipeptide repeat protein burden in C9orf72-associated ALS/FTD . Researchers can leverage RUVBL2 antibodies to further investigate this emerging area:

• Patient-derived cellular models:

  • Compare RUVBL2 levels between patient and control cells using validated antibodies

  • Analyze both protein (immunoblot) and transcript (RT-qPCR) levels as discrepancies have been reported

  • Establish correlations between RUVBL2 expression and disease phenotypes

• Mechanistic investigation approaches:

  • Use HRP-conjugated RUVBL2 antibodies for high-sensitivity detection in limited patient samples

  • Implement co-localization studies with dipeptide repeat proteins and stress granule markers

  • Assess RUVBL2 distribution changes in response to cellular stress conditions relevant to neurodegeneration

• Therapeutic exploration strategies:

  • Monitor RUVBL2 levels as a potential biomarker during experimental therapeutic interventions

  • Consider RUVBL2 modulation as a therapeutic approach based on its protective role against toxic protein aggregation

  • Develop cell-based screening assays using RUVBL2 antibodies to identify compounds that enhance its protective function

• Technical considerations:

  • Include appropriate loading controls (GAPDH has been validated)

  • Ensure antibody specificity in neuronal contexts

  • Consider post-translational modifications that may be tissue-specific

What are the emerging applications of RUVBL2 antibodies in cancer research?

RUVBL2 dysregulation has been implicated in various cancers, making this an important area for antibody-based research:

• Expression analysis in tumor samples:

  • Use immunohistochemistry with optimized protocols (recommended dilution 1:250-1:1000)

  • Develop tissue microarray approaches for high-throughput analysis

  • Correlate expression patterns with clinical outcomes and molecular subtypes

• Functional investigations:

  • Study RUVBL2's role in cancer-associated transcriptional programs (e.g., c-Myc)

  • Investigate its contribution to DNA damage response pathways in cancer cells

  • Explore connections to chromatin remodeling in oncogene-driven transcriptional reprogramming

• Therapeutic target validation:

  • Use antibodies to confirm target engagement in drug development

  • Monitor RUVBL2 complex formation as a pharmacodynamic marker

  • Develop proximity-based assays to screen for molecules disrupting critical RUVBL2 interactions

• Methodological considerations:

  • Validate antibodies across diverse cancer cell lines before clinical sample analysis

  • Consider tumor heterogeneity in sampling approaches

  • Implement multiplexed detection systems to correlate RUVBL2 with established cancer biomarkers