RUVBL2 Antibody

What is RUVBL2 and what cellular functions make it an important research target?

RUVBL2 (RuvB-like 2) is a highly conserved member of the AAA+ (ATPases Associated with various cellular Activities) protein subfamily characterized by Walker A and B motifs involved in ATP binding and hydrolysis. The protein possesses single-stranded DNA-stimulated ATPase and ATP-dependent DNA helicase (5' to 3') activity . RUVBL2 functions as a component of several critical multi-protein complexes including:

• The NuA4 histone acetyltransferase complex, which modifies nucleosomal histones H4 and H2A

• SWR1-like complexes that regulate histone exchange

• The TIP60 acetyltransferase complex involved in DNA damage response

• Telomerase biogenesis machinery

RUVBL2 plays essential roles in transcriptional regulation, chromatin remodeling, DNA repair, and oncogenic transformation through its interaction with MYC and modulation of LEF1/TCF1-CTNNB1 complex activity . Its involvement in these fundamental cellular processes makes it a significant target for both basic research and disease-focused investigations.

What are the common aliases and identifiers for RUVBL2 protein?

RUVBL2 is known by numerous aliases in scientific literature, which can create challenges when conducting comprehensive research:

When searching literature or antibody databases, researchers should use multiple name variations to ensure comprehensive results . RUVBL2's involvement in multiple protein complexes also explains its importance across diverse cellular functions.

What types of RUVBL2 antibodies are available for research applications?

Researchers can choose from several types of RUVBL2 antibodies based on their specific experimental requirements:

Polyclonal antibodies:

• Typically rabbit-derived (e.g., 10195-1-AP, NBP1-33599)

• Generated against various immunogens including:

  • Full recombinant proteins

  • N-terminal region peptides

  • Central domain sequences

• Purified via antigen affinity or Protein A methods

• Offer broader epitope recognition

Monoclonal antibodies:

• Mouse-derived options (e.g., 67851-1-Ig, OTI2B9 clone)

• Targeted to specific epitopes such as amino acids 113-370

• Provide highly consistent lot-to-lot reproducibility

• Available in various isotypes (IgG2b common)

Each antibody type offers distinct advantages: polyclonals provide robust detection across multiple epitopes while monoclonals offer higher specificity for particular regions of RUVBL2. The choice depends on experimental requirements and whether specific RUVBL2 domains or complexes are being investigated .

What species reactivity is expected with RUVBL2 antibodies?

RUVBL2 shows high sequence conservation across species, enabling many antibodies to cross-react with orthologs from multiple organisms:

How should researchers optimize Western blot conditions for RUVBL2 detection?

Western blot optimization for RUVBL2 detection requires attention to several key parameters:

Antibody dilution:
Different antibodies require specific dilutions for optimal results:

• Polyclonal antibodies: 1:500-1:3000 range typical

• Monoclonal antibodies: Can range from 1:1000 to 1:50000 (extremely sensitive)

Sample preparation:

• Expected molecular weight: 51 kDa

• Positive controls: HeLa, HepG2, A549, Jurkat cell lysates

• Loading recommendation: 20-35 μg total protein per lane

• Denaturation: Complete denaturation critical for exposing RUVBL2 epitopes

Detection optimization:

• Signal enhancement: When using less sensitive detection methods, consider longer primary antibody incubation (overnight at 4°C)

• Blocking: 5% non-fat milk or BSA in TBS-T typically effective

• Membrane washing: Thorough washing critical to reduce background

Different RUVBL2 antibodies may detect slightly different bands based on their epitope recognition and potential post-translational modifications of RUVBL2 .

What conditions are recommended for immunohistochemical detection of RUVBL2?

For successful immunohistochemical (IHC) detection of RUVBL2 in tissues:

Antigen retrieval:

• Primary recommendation: Tris-EDTA (TE) buffer at pH 9.0

• Alternative method: Citrate buffer at pH 6.0

• Specific protocol: For testicular tissue, Trilogy™ (EDTA-based, pH 8.0) buffer with 15-minute retrieval

Antibody dilutions:

• Polyclonal antibodies: 1:250-1:2000 range

• Monoclonal antibodies: 1:500-1:2000 range

• Titration recommended for each tissue type

Validated tissue samples:

• Positive controls: Human lung cancer, gliomas, placenta

• Animal tissues: Mouse/rat bladder, mouse testis

• Nuclear and sometimes cytoplasmic staining patterns expected

Proper controls and optimization of staining conditions are essential for accurate interpretation of RUVBL2 expression patterns in different tissues and pathological conditions.

What procedures are essential for successful immunofluorescence detection of RUVBL2?

For optimal immunofluorescence (IF) detection of RUVBL2:

Fixation and permeabilization:

• Paraformaldehyde fixation (4%, 10-15 minutes) validated for most cell lines

• Permeabilization with 0.1-0.3% Triton X-100 critical for nuclear protein access

• Alternative fixation: Cold methanol (10 min) may better expose certain epitopes

Antibody parameters:

• Recommended dilutions: 1:200-1:800 range for most antibodies

• Incubation conditions: 1-2 hours at room temperature or overnight at 4°C

• Secondary antibody selection: Match host species; highly cross-adsorbed versions reduce background

Validated cell lines:

• HeLa cells

• HepG2 cells

• A549 cells

RUVBL2 typically shows predominantly nuclear localization with possible nucleolar enrichment, though some cytoplasmic staining may also be observed depending on cell type and physiological state .

How can researchers effectively perform immunoprecipitation with RUVBL2 antibodies?

For successful immunoprecipitation (IP) of RUVBL2 and associated complexes:

Antibody amounts:

• Typical range: 0.5-4.0 μg antibody per 1.0-3.0 mg total protein lysate

• May require titration for specific applications and antibodies

Validated sources:

• HeLa cells

• Mouse brain tissue

• Other cell lines expressing RUVBL2

Protocol considerations:

• Lysis buffer: Use buffers that maintain protein-protein interactions if studying complexes (e.g., NP-40 or CHAPS-based)

• Pre-clearing: Recommended to reduce non-specific binding

• Controls: Include IgG control from same species as antibody

• Elution: Gentle conditions if maintaining complex integrity is important

• Verification: Western blot with a different RUVBL2 antibody (different epitope/species)

IP applications are particularly valuable for studying RUVBL2's interactions with RUVBL1 and other complex components involved in chromatin remodeling and transcriptional regulation.

How can RUVBL2 autoantibodies be detected and what is their clinical significance?

RUVBL2 autoantibodies have been identified as novel biomarkers in systemic sclerosis (SSc):

Detection methods:

• Protein immunoprecipitation assays detecting ~50 kDa doublets

• RNA immunoprecipitation complementary analysis

• Liquid chromatography-mass spectrometry of affinity-purified autoantigens

• Immunoblot confirmation assays

Clinical associations:

• Prevalence: Detected in 1.1-1.9% of SSc patients across multiple cohorts

• Exclusivity: Not detected in disease controls or healthy subjects

• Clinical features: Associated with:

  • Higher frequency of SSc-myositis overlap

  • Diffuse cutaneous involvement

  • Older age at SSc onset

  • Predominantly male patients

Distinguishing characteristics:
Compared to other SSc/myositis overlap autoantibodies (anti-PM-Scl and anti-Ku), anti-RUVBL1/2 shows distinctive demographic and clinical associations, suggesting it defines a unique subset of SSc patients .

This represents an important translational application where research antibodies enabled the discovery of diagnostic autoantibodies that may have clinical utility.

What techniques can be employed to study RUVBL2's ATPase and helicase activities?

RUVBL2's enzymatic activities can be assessed through several sophisticated biochemical approaches:

ATPase activity assays:

• Malachite green phosphate detection assay

• Coupled enzyme systems using pyruvate kinase/lactate dehydrogenase

• ADP-Glo™ luminescent detection

• Radioactive [γ-32P]ATP hydrolysis measurement

Helicase activity assessment:

• DNA substrate design: Partially complementary oligonucleotides with fluorescent labels

• Detection methods: Gel-based separation of unwound substrates

• Direction confirmation: RUVBL2 shows 5' to 3' directional activity

• Stimulation factors: Single-stranded DNA enhances ATPase activity

Experimental considerations:

• Buffer conditions: Require Mg2+ for activity

• Temperature: Typically assayed at 37°C

• ATP concentration: Usually 1-5 mM range

• Control reactions: Include ATP-binding site mutants (Walker A mutations)

These biochemical assays provide critical insights into how RUVBL2 contributes functionally to chromatin remodeling complexes and DNA repair mechanisms .

How can researchers study RUVBL1/RUVBL2 complex formation and function?

The RUVBL1/RUVBL2 complex forms a functionally important structure that can be studied through multiple approaches:

Complex isolation methods:

• Co-immunoprecipitation using antibodies against either protein

• Tandem affinity purification with tagged versions

• Size-exclusion chromatography to isolate native complexes

• Blue native PAGE to analyze intact complexes

Structural analysis approaches:

• Cryo-electron microscopy of purified complexes

• X-ray crystallography of recombinant proteins

• Crosslinking mass spectrometry to map interaction interfaces

• Hydrogen-deuterium exchange mass spectrometry

Functional analysis:

• ATPase and helicase assays of isolated complexes vs. individual proteins

• Reconstitution experiments with wild-type and mutant subunits

• Cellular localization studies using dual immunofluorescence

• ChIP-seq to identify genomic binding sites

In vivo interaction detection:

• Proximity ligation assay (PLA) for visualizing interactions in situ

• FRET/BRET for measuring protein-protein proximity

• Split reporter complementation assays

Understanding the RUVBL1/RUVBL2 complex provides insights into how these proteins function cooperatively in chromatin remodeling and transcriptional regulation .

What approaches can reveal RUVBL2's role in transcriptional regulation?

RUVBL2's involvement in transcriptional regulation can be investigated through various molecular and cellular techniques:

Chromatin association studies:

• Chromatin immunoprecipitation (ChIP) followed by qPCR or sequencing

• CUT&RUN or CUT&Tag for higher resolution chromatin binding profiles

• Re-ChIP to identify co-occupancy with transcription factors

Transcriptional output analysis:

• RNA-seq after RUVBL2 knockdown/knockout

• Nascent RNA analysis (e.g., GRO-seq, NET-seq)

• Reporter gene assays for specific promoters

• Nuclear run-on assays

Protein interaction mapping:

• Mass spectrometry identification of RUVBL2-associated transcription factors

• Co-IP with known transcriptional regulators (e.g., MYC, LEF1/TCF1-CTNNB1)

• Yeast two-hybrid or mammalian two-hybrid screening

Chromatin structure analysis:

• ATAC-seq to assess chromatin accessibility changes

• MNase-seq for nucleosome positioning

• Hi-C to examine higher-order chromatin structure

These approaches have revealed RUVBL2's essential roles in oncogenic transformation by MYC, modulation of TCF1-CTNNB1 complex activity, and inhibition of ATF2 transcriptional activity .

How does RUVBL2 contribute to DNA damage repair mechanisms?

RUVBL2's involvement in DNA damage repair can be investigated through specialized techniques:

Damage response localization:

• Immunofluorescence to track RUVBL2 recruitment to DNA damage sites

• Laser microirradiation combined with live-cell imaging

• Proximity labeling at damage sites

• ChIP analysis of damage-induced binding

Repair pathway assessment:

• Homologous recombination reporter assays

• Non-homologous end joining (NHEJ) assays

• Single-strand annealing assessment

• DNA damage sensitivity assays after RUVBL2 depletion

Complex participation analysis:

• Co-IP with DNA repair proteins after damage induction

• RUVBL2 recruitment to TIP60 complex following damage

• Analysis of RUVBL2's contribution to NuA4 complex DNA repair functions

Enzymatic activity contribution:

• ATPase activity requirements for repair

• Helicase activity contribution to damage processing

• Mutational analysis of catalytic domains

RUVBL2's evolutionary relationship to bacterial RuvB (essential for homologous recombination and double-strand break repair) suggests conservation of crucial DNA repair functions throughout evolution .

What strategies can address common challenges in RUVBL2 Western blotting?

Researchers frequently encounter specific challenges when detecting RUVBL2 by Western blot:

Problem: High background
Solutions:

• Optimize blocking (5% BSA may be superior to milk for some antibodies)

• Increase washing duration and number of washes

• Dilute antibody further (particularly for high-sensitivity antibodies like 67851-1-Ig that can be used at 1:50000)

• Use newer membrane technologies with lower background properties

Problem: Weak signal
Solutions:

• Use validated positive controls (HeLa, HepG2, A549 cells)

• Ensure complete protein transfer (verify with reversible stain)

• Consider longer primary antibody incubation (overnight at 4°C)

• Use highly sensitive detection reagents

• Load more total protein (35-50 μg)

Problem: Multiple bands
Solutions:

• Include fresh protease inhibitors in lysis buffer

• Verify sample integrity with other protein targets

• Consider deglycosylation treatment if glycosylation is suspected

• Compare results with multiple RUVBL2 antibodies recognizing different epitopes

Systematic optimization of these parameters typically resolves most Western blotting issues for RUVBL2 detection .

How can immunohistochemical staining for RUVBL2 be improved in challenging tissues?

For optimizing RUVBL2 immunohistochemistry in difficult tissue samples:

Antigen retrieval optimization:

• Test both recommended methods (TE buffer pH 9.0 and citrate buffer pH 6.0)

• Adjust retrieval duration (15-30 minutes)

• Try pressure cooker vs. microwave vs. water bath methods

• For mouse testis specifically, Trilogy™ buffer (EDTA-based, pH 8.0) with 15-minute retrieval has proven effective

Signal amplification options:

• Polymer detection systems offer superior sensitivity

• Tyramide signal amplification for very low abundance targets

• ABC (avidin-biotin complex) method as an alternative approach

• Extended chromogen development for weakly expressing tissues

Background reduction:

• Include avidin/biotin blocking for tissues with endogenous biotin

• Use specific blocking serums matched to secondary antibody

• Add protein blocking steps (1% BSA, 10% normal serum)

• Optimize primary antibody concentration (1:250-1:1000 range recommended)

Each tissue type may require specific optimization due to differences in fixation, processing, and endogenous protein levels .

What controls should be included in RUVBL2 antibody experiments?

Proper experimental controls are essential for reliable interpretation of RUVBL2 antibody results:

Positive controls:

• Cell lysates: HeLa, HepG2, A549, Jurkat, K-562 cells

• Tissues: Human lung cancer, gliomas, placenta tissue

• Animal tissues: Mouse/rat bladder, mouse testis

Negative controls:

• Antibody controls: Isotype-matched non-specific IgG

• Secondary-only controls (omit primary antibody)

• Absorption controls (pre-incubation of antibody with immunizing peptide)

Validation controls:

• RUVBL2 knockdown/knockout cells (multiple publications demonstrate use)

• Overexpression systems (shown in flow cytometry validation)

• Multiple antibodies targeting different epitopes

• Recombinant RUVBL2 protein as Western blot standard

Application-specific controls:

• For IP: IgG pull-down control

• For IF: Subcellular markers to confirm localization

• For IHC: Normal adjacent tissue controls

These comprehensive controls help ensure specificity of detection and accurate interpretation of experimental results across different applications .

How can RUVBL2 expression be measured in cancer research applications?

Cancer researchers can assess RUVBL2 expression through multiple complementary techniques:

Protein level analysis:

• Immunohistochemistry on tissue microarrays

• Western blotting of tumor vs. normal tissue

• Reverse phase protein arrays for high-throughput screening

• Flow cytometry for cell-by-cell quantification

Transcript analysis:

• RT-qPCR for RUVBL2 mRNA quantification

• RNA-seq for transcriptome-wide context

• In situ hybridization for spatial expression patterns

• NanoString assays for fixed tissue samples

Clinical sample validation:

• Human lung cancer tissue has been validated for RUVBL2 IHC

• Human gliomas tissue shows RUVBL2 expression

• Multiple cancer cell lines express detectable RUVBL2

Functional significance:
RUVBL2 plays essential roles in oncogenic transformation by MYC and modulates LEF1/TCF1-CTNNB1 complex activity, potentially contributing to cancer progression . These connections make RUVBL2 a target of interest in cancer biology research.

What methodologies can reveal RUVBL2's interaction with chromatin remodeling complexes?

RUVBL2's participation in chromatin remodeling complexes can be investigated using specialized approaches:

Complex isolation methods:

• Tandem affinity purification of RUVBL2-containing complexes

• Glycerol gradient ultracentrifugation for complex separation

• Ion exchange chromatography followed by immunoblotting

• Size exclusion chromatography to separate intact complexes

Chromatin association analysis:

• ChIP-seq to map genomic binding sites

• CUT&RUN for higher resolution and lower background

• ChIP-MS to identify co-bound factors

• Sequential ChIP to determine complex co-occupancy

Functional genomics approaches:

• CRISPR-Cas9 targeting of RUVBL2 followed by ATAC-seq

• Auxin-inducible degron systems for rapid RUVBL2 depletion

• Targeted protein degradation approaches

• Specific domain mutations to disrupt complex formation

Histone modification analysis:

• ChIP-seq for histone acetylation changes (H4 and H2A)

• Mass spectrometry of histone post-translational modifications

• In vitro histone modification assays with reconstituted complexes

These approaches have revealed RUVBL2's participation in NuA4 histone acetyltransferase complex, SWR1-like complexes mediating histone H2A.Z removal, and INO80 complex functions .

What new research directions are emerging for RUVBL2 in disease models?

Recent findings have opened several promising research directions for RUVBL2 in disease contexts:

Neurodegenerative conditions:

• RUVBL2 has been associated with neuronopathy and distal hereditary motor neuronopathy

• Potential research focus on DNA repair defects in neuronal cells

• Mouse models with conditional RUVBL2 knockout in neural tissues

Systemic sclerosis biomarkers:

• Anti-RUVBL1/2 autoantibodies define a unique SSc subset with myositis overlap

• Development of diagnostic assays for clinical application

• Investigation of mechanisms triggering autoimmunity against RUVBL2

Cancer therapeutic targeting:

• Exploration of RUVBL2 ATPase inhibitors

• Disruption of RUVBL2-dependent transcriptional programs

• Synthetic lethality approaches in MYC-driven cancers

• Investigation of differential requirements in cancer vs. normal cells

Developmental biology:

• RUVBL2's role in embryonic development

• Tissue-specific functions in stem cell maintenance

• Interactions with developmental signaling pathways

These emerging research directions highlight RUVBL2's significance across diverse biological processes and disease mechanisms , making it a promising target for translational research.