RUVBL2 Antibody, FITC conjugated

What is RUVBL2 and what are its primary cellular functions?

RUVBL2 (RuvB-like 2) is an essential AAA+ ATPase that functions as a co-chaperone in various cellular processes. It possesses single-stranded DNA-stimulated ATPase and ATP-dependent DNA helicase (5' to 3') activity. Hexamerization of RUVBL2 is critical for ATP hydrolysis, with adjacent subunits in the ring-like structure contributing to the ATPase activity. RUVBL2 plays essential roles in transcriptional regulation, DNA replication, and as a component of several multiprotein complexes, including the PAQosome/R2TP complex. In transcriptional regulation, RUVBL2 enhances the co-phase separation of RPB1 CTD and transcription factors, directly regulating RNA Polymerase II clustering and transcription activation .

How does RUVBL2 interact with the transcriptional machinery?

RUVBL2 predominantly interacts with the unphosphorylated C-terminal domain (CTD) of RNA Polymerase II's largest subunit (RPB1) on chromatin. This interaction differs from its role in the R2TP complex, where it helps assemble the Pol II complex through the RPB5 subunit in the cytoplasm. RUVBL2 localizes to active transcription sites, and tethered wild-type RUVBL2 is associated with enhanced Pol II signal intensity (61% increase) at experimental loci when gene activation occurs. This indicates that RUVBL2 promotes Pol II clustering during transcription activation, without significantly changing the nuclear Pol II levels, suggesting a specific role in Pol II organization rather than assembly .

What protein complexes involve RUVBL2 and what are their functions?

RUVBL2 participates in several critical protein complexes:

• PAQosome/R2TP complex: Involved in the assembly of RNA Polymerase II in the cytoplasm

• INO80 chromatin remodeling complex: As the INO80J subunit

• RUVBL1/RUVBL2 heterohexamers or heterododecamers: Function in various cellular contexts

RUVBL2's ATPase activity is essential for the maturation or dissociation of the PAQosome complex. When this activity is inhibited, PAQosome components show increased interaction with RUVBL2, suggesting the complex can assemble but is unable to properly mature or dissociate. This affects the stability of PAQosome client proteins, including members of the PIKK family .

What applications are optimal for FITC-conjugated RUVBL2 antibodies?

FITC-conjugated RUVBL2 antibodies are particularly valuable for fluorescence-based detection methods, including:

• Flow cytometry: For quantifying RUVBL2 expression in cell populations

• Immunofluorescence microscopy: For visualizing RUVBL2 subcellular localization

• ELISA: For quantitative detection of RUVBL2 in solution

• Live-cell imaging: For tracking RUVBL2 dynamics in real-time

The direct FITC conjugation eliminates the need for secondary antibodies, reducing background and cross-reactivity issues while streamlining experimental workflows. When studying RUVBL2's association with transcriptional activation sites or its recruitment to specific chromatin regions, the FITC conjugate enables direct visualization of these interactions .

How should RUVBL2 antibody specificity be validated for chromatin immunoprecipitation experiments?

Validating RUVBL2 antibody specificity for ChIP experiments should involve multiple approaches:

• Western blot verification: Confirm a single band at the expected molecular weight (~51 kDa)

• Knockdown/knockout controls: Compare ChIP results from RUVBL2-depleted cells to verify signal specificity

• Peptide competition assays: Pre-incubate antibody with the immunogen peptide (RUVBL2 281-444AA fragment) to confirm specific blocking of signal

• IP-Western validation: Perform immunoprecipitation followed by Western blot detection with a different RUVBL2 antibody

• ChIP-qPCR at known RUVBL2-bound loci: Test enrichment at established targets versus negative control regions

This validation is particularly important since RUVBL2 forms complexes with RUVBL1 and other proteins, and the antibody must specifically recognize RUVBL2 within these complexes on chromatin .

What buffer conditions optimize FITC-conjugated RUVBL2 antibody performance?

Optimal buffer conditions for FITC-conjugated RUVBL2 antibody include:

Avoid repeated freeze-thaw cycles as this can damage both the antibody and the FITC conjugate. Store at -20°C or -80°C in aliquots. Note that exposure to strong light can photobleach the FITC fluorophore, so samples should be protected from light during storage and experiments .

How can researchers differentiate between RUVBL2's transcriptional versus DNA replication functions in experimental data?

Distinguishing between RUVBL2's transcriptional and DNA replication functions requires careful experimental design:

• Cell cycle synchronization: Compare RUVBL2 chromatin association patterns between synchronized cells in G1 (predominantly transcriptional) versus S phase (DNA replication)

• Co-localization analysis: Use dual-color imaging with markers specific to:

  • Transcription: Unphosphorylated RNA Pol II, active transcription site markers

  • Replication: PCNA, BrdU incorporation, or other replication fork markers

• Selective inhibition: Use transcription inhibitors (e.g., α-amanitin) or replication inhibitors (e.g., aphidicolin) to selectively block one process

• Domain-specific mutants: Generate RUVBL2 mutants that selectively disrupt interaction with transcription versus replication machinery

• ChIP-seq versus repli-seq comparison: Map RUVBL2 binding sites and compare with active transcription sites versus replication origins

These approaches can help resolve whether observed phenotypes after RUVBL2 manipulation stem from its transcriptional regulation or DNA replication functions .

What are common pitfalls in RUVBL2 antibody-based experiments and how can they be addressed?

Common pitfalls and their solutions include:

• Cross-reactivity with RUVBL1: Due to structural similarity (65-70% sequence homology)

  • Solution: Validate with RUVBL1 knockdown cells; use antibodies raised against unique regions

• Detection of different oligomeric states:

  • Solution: Use native PAGE conditions to preserve complexes; include controls for monomeric versus oligomeric forms

• Epitope masking in protein complexes:

  • Solution: Test multiple antibodies targeting different RUVBL2 epitopes; use mild detergents to partially expose epitopes

• FITC photobleaching:

  • Solution: Minimize light exposure; use anti-fade mounting media; consider signal intensifiers or image immediately

• Interference from endogenous biotin when using streptavidin systems:

  • Solution: Include biotin blocking steps in protocols involving avidin-biotin detection systems

Careful controls and method optimization can help minimize these issues and improve data reliability .

How should researchers interpret conflicting data between RUVBL2 antibody detection and functional assays?

When faced with discrepancies between antibody-based detection and functional assays:

• Consider epitope accessibility: RUVBL2's conformation changes based on ATP binding state, potentially affecting epitope exposure

  • Test alternative antibodies targeting different epitopes

  • Compare fixed versus non-fixed samples

• Evaluate complex formation interference:

  • RUVBL2's incorporation into different complexes (R2TP, PAQosome, INO80) may mask antibody binding sites

  • Use biochemical fractionation to separate different complexes before analysis

• Assess post-translational modifications:

  • Phosphorylation or other modifications may affect antibody recognition

  • Use phosphatase treatment to determine if modifications impact detection

• Validate assay specificity:

  • Use CRISPR/Cas9 knockout cells as negative controls

  • Perform rescue experiments with wild-type versus mutant RUVBL2

• Examine subcellular localization discrepancies:

  • RUVBL2 functions differently in chromatin, nucleoplasm, and cytoplasm

  • Use cellular fractionation to resolve compartment-specific activities

Comprehensive validation across multiple experimental approaches can help resolve such conflicting data .

How can researchers investigate RUVBL2's role in phase separation and transcriptional condensates?

To study RUVBL2's role in phase separation and transcriptional condensates:

• Fluorescence recovery after photobleaching (FRAP):

  • Tag RUVBL2 with fluorescent proteins

  • Measure dynamics within condensates versus diffuse nuclear pool

  • Compare wild-type RUVBL2 versus ATPase-deficient mutants

• Optogenetic approaches:

  • Create light-inducible RUVBL2 clustering systems

  • Observe consequences on Pol II clustering and transcriptional activation

• In vitro phase separation assays:

  • Purify recombinant RUVBL2 and RPB1 CTD

  • Test direct effects on phase separation under various conditions

  • Analyze how ATP hydrolysis affects condensate dynamics

• Super-resolution microscopy:

  • Visualize nanoscale organization of RUVBL2 within transcriptional condensates

  • Perform multi-color imaging with Pol II and other transcription factors

• Proximity labeling (BioID/APEX):

  • Map the protein neighborhood of RUVBL2 within condensates

  • Compare composition between active and inactive transcriptional states

These approaches can reveal mechanistic insights into how RUVBL2 enhances co-phase separation of RPB1 CTD and transcription factors to regulate gene expression .

What methodologies can differentiate between RUVBL2's R2TP-dependent and independent functions?

To distinguish between R2TP-dependent and independent functions of RUVBL2:

• Selective protein depletion strategies:

  • Deplete R2TP-specific components (RPAP3, PIH1D1) versus RUVBL2 alone

  • Compare phenotypic consequences and molecular signatures

• Domain-specific mutations:

  • Generate RUVBL2 mutants that disrupt specific protein-protein interactions

  • Create mutations that affect ATPase activity but preserve structural integrity

• Cellular fractionation combined with proteomics:

  • Compare RUVBL2-associated proteins in cytoplasmic versus chromatin fractions

  • Identify differential interactomes that correspond to R2TP versus other functions

• Sequential ChIP (Re-ChIP):

  • Perform first ChIP with RUVBL2 antibody followed by second ChIP with other complex components

  • Map genomic sites where RUVBL2 functions with versus without R2TP complex

• Rapid protein degradation systems:

  • Use auxin-inducible or dTAG degron systems for selective, rapid depletion

  • Analyze immediate versus delayed consequences to differentiate direct from indirect effects

These approaches can help determine which cellular functions depend on RUVBL2 as part of the R2TP complex versus its roles in other complexes or as part of RUVBL1/RUVBL2 heterohexamers .

How can advanced imaging approaches be optimized for tracking RUVBL2 dynamics during transcriptional activation?

Advanced imaging approaches for tracking RUVBL2 dynamics during transcriptional activation:

• Live-cell imaging optimization:

  • Use bright, photostable fluorophores (consider Janelia Fluor dyes if direct conjugation is possible)

  • Implement lattice light-sheet microscopy for reduced phototoxicity and improved temporal resolution

  • Develop CRISPR knock-in cell lines expressing minimally tagged RUVBL2 at endogenous levels

• Single-molecule tracking:

  • Apply stochastic optical reconstruction microscopy (STORM) or photoactivated localization microscopy (PALM)

  • Track individual RUVBL2 molecules to measure diffusion rates, residence times, and clustering behavior

  • Compare dynamics at active versus inactive transcription sites

• Fluorescence correlation spectroscopy (FCS):

  • Measure diffusion coefficients to detect changes in complex size/composition

  • Analyze fluctuations before and after transcriptional stimulation

• Förster resonance energy transfer (FRET):

  • Design FRET pairs between RUVBL2 and Pol II or other transcription factors

  • Measure interaction dynamics during transcriptional activation in real-time

• Correlative light and electron microscopy (CLEM):

  • Combine fluorescence imaging of RUVBL2-FITC with ultrastructural analysis

  • Visualize RUVBL2 localization relative to transcription factories at nanometer resolution

These advanced imaging approaches can provide unprecedented insights into the spatiotemporal dynamics of RUVBL2 during transcriptional regulation processes .

What assays can quantify RUVBL2 ATPase activity in different cellular contexts?

Several complementary approaches can quantify RUVBL2 ATPase activity:

• Malachite green phosphate assay:

  • Purify RUVBL2 complexes (alone or with interacting partners)

  • Measure inorganic phosphate release from ATP hydrolysis

  • Compare activity with different stimulators (ssDNA, protein partners)

• Bioluminescent ADP detection:

  • Use luciferase-based assays to measure ADP production

  • Higher sensitivity for detecting subtle changes in ATPase activity

  • Compatible with high-throughput screening formats

• FRET-based ATP sensors:

  • Express genetically-encoded ATP sensors in cells

  • Monitor local ATP consumption in RUVBL2-enriched regions

  • Measure real-time changes during transcriptional activation

• ATPase-inactive mutants:

  • Generate Walker A/B motif mutations (K83A, E133Q)

  • Use as controls to validate assay specificity

  • Compare phenotypic effects versus wild-type RUVBL2

• In-gel ATPase assays:

  • Separate native complexes by non-denaturing PAGE

  • Perform in-gel ATP hydrolysis with lead phosphate precipitation

  • Identify which specific RUVBL2-containing complexes possess ATPase activity

This multi-method approach can distinguish the ATPase activity of RUVBL2 in different cellular contexts and protein complexes .

How can researchers investigate the relationship between RUVBL2 ATPase activity and PAQosome maturation?

To investigate the relationship between RUVBL2 ATPase activity and PAQosome maturation:

• Time-course analysis with ATPase inhibitors:

  • Treat cells with specific RUVBL2 ATPase inhibitors

  • Monitor PAQosome assembly versus maturation/dissociation using co-immunoprecipitation

  • Track post-translational modifications associated with complex maturation

• Structure-function studies:

  • Introduce mutations at RUVBL1/RUVBL2 interfaces that affect ATP binding/hydrolysis

  • Assess effects on PAQosome client protein stability

  • Perform structure-guided mutagenesis of regions identified in resistance-conferring mutations

• Single-particle cryo-EM:

  • Compare PAQosome structures with wild-type versus ATPase-dead RUVBL2

  • Capture intermediate states during complex maturation

  • Visualize conformational changes dependent on nucleotide-bound state

• Client protein handoff assays:

  • Develop FRET-based systems to monitor transfer of client proteins from PAQosome

  • Determine how ATPase activity influences client release kinetics

  • Test whether different client proteins have different dependencies on ATPase activity

• Proteomic profiling of PAQosome dynamics:

  • Use quantitative mass spectrometry to track complex composition over time

  • Compare protein interaction networks with and without ATPase inhibition

  • Identify key transition points dependent on ATP hydrolysis

These approaches can reveal how RUVBL2 ATPase activity drives the functional cycle of PAQosome assembly, maturation, and client protein processing .

What approaches can resolve contradictory findings about RUVBL2's role in gene expression?

To resolve contradictory findings about RUVBL2's role in gene expression:

• Cell type-specific analysis:

  • Compare RUVBL2 functions across different cell types

  • Correlate with expression levels of interacting partners

  • Consider developmental stage and differentiation status

• Genome-wide versus locus-specific effects:

  • Combine ChIP-seq with RNA-seq after RUVBL2 perturbation

  • Distinguish direct versus indirect transcriptional effects

  • Use CUT&RUN or CUT&Tag for higher resolution chromatin mapping

• Temporal dynamics considerations:

  • Implement rapid protein depletion systems (AID, dTAG)

  • Distinguish immediate versus adaptive transcriptional responses

  • Perform time-series experiments after RUVBL2 perturbation

• Context-dependent complexes:

  • Map RUVBL2-containing complexes at different gene regulatory elements

  • Compare RUVBL2 function at enhancers versus promoters

  • Assess whether RUVBL2 has activating or repressive roles depending on complex formation

• Integration with epigenetic landscape:

  • Correlate RUVBL2 binding with histone modifications

  • Examine relationships with chromatin accessibility

  • Investigate interactions with other chromatin-modifying complexes

This systematic approach can help reconcile seemingly contradictory findings by revealing context-dependent functions of RUVBL2 in gene expression regulation .

How should the dual nuclear and cytoplasmic functions of RUVBL2 be experimentally distinguished?

To experimentally distinguish nuclear versus cytoplasmic functions of RUVBL2:

• Compartment-specific protein targeting:

  • Create fusion proteins with additional nuclear localization signals (NLS) or nuclear export signals (NES)

  • Test whether forced localization rescues specific phenotypes after RUVBL2 depletion

  • Use rapamycin-inducible dimerization systems for dynamic translocation control

• Selective immunoprecipitation from distinct fractions:

  • Perform separate IP-MS analyses from purified nuclear, chromatin, and cytoplasmic fractions

  • Compare interaction networks and complex composition

  • Identify compartment-specific post-translational modifications

• Domain-specific mutations with localization consequences:

  • Map domains required for nuclear import/export

  • Generate mutants that alter subcellular distribution without affecting catalytic activity

  • Assess which functions are restored by compartment-specific mutants

• Proximity labeling with compartment-specific anchors:

  • Use split BioID or APEX systems with one component restricted to specific compartments

  • Compare RUVBL2 neighborhoods in different cellular locations

  • Identify location-specific interaction partners

• Live-cell imaging with optogenetic control:

  • Develop light-inducible systems to sequester RUVBL2 in specific compartments

  • Monitor acute effects on nuclear versus cytoplasmic processes

  • Track compensation mechanisms over time

These approaches can dissect the distinct roles of RUVBL2 in the cytoplasm (e.g., R2TP-dependent Pol II assembly) versus nucleus (transcriptional regulation and DNA replication) .

How might novel RUVBL2 imaging tools advance our understanding of its function in disease contexts?

Advanced RUVBL2 imaging tools could transform disease research through:

• Patient-derived organoid imaging:

  • Track RUVBL2 dynamics in 3D cancer organoids

  • Correlate localization patterns with treatment response

  • Identify patient-specific alterations in RUVBL2 behavior

• Multiplexed imaging for precision medicine:

  • Develop antibody panels to simultaneously detect RUVBL2, its partners, and modification states

  • Create diagnostic signatures based on RUVBL2 complex formation patterns

  • Correlate with clinical outcomes in cancer and other diseases

• Intravital microscopy applications:

  • Track RUVBL2 dynamics in live animal models using surgical windows

  • Monitor responses to therapeutic interventions in real-time

  • Assess RUVBL2 behavior in tumor microenvironments

• Spatial transcriptomics integration:

  • Combine RUVBL2 imaging with spatial transcriptomics

  • Correlate RUVBL2 clustering with local gene expression patterns

  • Map functional consequences of RUVBL2 dysregulation in tissue context

• Machine learning image analysis:

  • Develop AI algorithms to identify subtle changes in RUVBL2 localization patterns

  • Predict disease progression based on RUVBL2 imaging features

  • Automate analysis of large-scale patient sample imaging data

These novel imaging approaches could reveal how RUVBL2 dysfunction contributes to diseases like cancer and identify new therapeutic opportunities targeting its activity or interactions .

What emerging technologies might revolutionize our understanding of RUVBL2 in transcriptional regulation?

Emerging technologies with potential to transform our understanding of RUVBL2 in transcription:

• Single-cell multi-omics:

  • Integrate single-cell ATAC-seq, RNA-seq, and proteomics

  • Correlate RUVBL2 levels with chromatin accessibility and gene expression

  • Map cell state transitions dependent on RUVBL2 activity

• Spatial genomics techniques:

  • Apply DNA MERFISH to visualize RUVBL2 binding sites in intact nuclei

  • Create 3D maps of RUVBL2 association with chromosomal territories

  • Correlate with transcriptional activity zones

• Base-resolution protein-DNA interaction mapping:

  • Implement CUT&Tag with enhanced base-pair resolution

  • Precisely map RUVBL2 positions relative to transcription start sites

  • Identify sequence-specific preferences in RUVBL2 chromatin association

• Phase separation biophysical tools:

  • Microrheology to measure physical properties of RUVBL2-containing condensates

  • Optogenetic control of condensate formation/dissolution

  • Selective perturbation of specific condensate components

• Genome-scale CRISPR functional screens:

  • Screen for genetic interactions that modify RUVBL2 transcriptional functions

  • Identify synthetic lethal relationships in cancer contexts

  • Map the genetic network surrounding RUVBL2-dependent processes