ripor2 Antibody

What is RIPOR2 and why is it important in cellular research?

RIPOR2 (also known as FAM65B, DFNA21, DFNB104) is a 118.5 kDa protein comprising 1068 amino acid residues that functions as an atypical inhibitor of the small GTPase RhoA . It is primarily localized in the cell membrane and cytoplasm, with significant expression in primary fetal mononuclear myoblasts . RIPOR2 plays critical roles in multiple cellular processes including myoblast fusion, hair cell differentiation, T lymphocyte proliferation, and neutrophil polarization . Its importance in research stems from its involvement in hearing mechanisms, immune function, and cancer progression, making it a valuable target for studying these physiological and pathological processes .

What types of RIPOR2 antibodies are available for research applications?

Several types of RIPOR2 antibodies are available for research use:

Most commercially available antibodies are polyclonal, derived from rabbit hosts, and demonstrate reactivity against human RIPOR2, with some exhibiting cross-reactivity with mouse and rat orthologs .

What are the best applications for RIPOR2 antibodies in basic research?

RIPOR2 antibodies are most commonly utilized in:

• Western Blotting (WB): Effective at dilutions of 1:500-1:2000 for detecting RIPOR2 protein in cell and tissue lysates

• Immunohistochemistry (IHC): Recommended dilutions of 1:20-1:200 for visualizing RIPOR2 in formalin-fixed, paraffin-embedded tissues

• Immunofluorescence (IF): Used at 1:50-1:200 dilutions to examine subcellular localization and translocation of RIPOR2

• ELISA: For quantitative detection and immunoprecipitation experiments

Selection of the appropriate application depends on whether you're investigating expression levels, localization patterns, or protein-protein interactions involving RIPOR2 .

How can I optimize RIPOR2 antibody-based immunoprecipitation for identifying novel interaction partners?

To optimize RIPOR2 antibody-based immunoprecipitation:

• Antibody selection: Use antibodies raised against different epitopes of RIPOR2 to ensure complete coverage. For example, antibodies targeting AA 1-250 region versus those targeting other domains may reveal different interaction partners .

• Cross-linking strategy: Implement reversible cross-linking with DSP (dithiobis(succinimidyl propionate)) at 0.5-2 mM for 30 minutes before cell lysis to capture transient interactions, as demonstrated in studies identifying RIPOR2's interaction with gentamicin (GEN) .

• Buffer optimization: For membrane-associated interactions, use buffers containing 1% NP-40 or 0.5% Triton X-100; for cytoskeletal interactions, include 0.5 mM MgCl₂ and reduce detergent concentration to 0.1% .

• Validation controls: Always include IgG control, lysate-only control, and for suspected interactions, competitive blocking with purified recombinant RIPOR2 protein .

• Confirmation strategy: Verify interactions through reciprocal co-immunoprecipitation and direct binding assays using purified proteins .

This approach has successfully identified novel interactions between RIPOR2 and proteins involved in autophagy pathways and aminoglycoside-induced ototoxicity .

What are the optimal protocols for studying RIPOR2 translocation in response to stimuli?

For studying RIPOR2 translocation:

• Time-course design: Implement short time intervals (2.5, 5, 15, 30, 60, and 120 minutes) after stimulation, as RIPOR2 has been shown to translocate from stereocilia base to pericuticular areas within 2.5 minutes of gentamicin exposure in cochlear hair cells .

• Fixation method: Use 4% paraformaldehyde for 20 minutes at room temperature, followed by permeabilization with 0.2% Triton X-100 for 10 minutes to preserve translocation events .

• Dual immunofluorescence: Combine RIPOR2 antibody (1:150 dilution) with markers for subcellular compartments:

  • Alexa-647 secondary antibodies for RIPOR2

  • Phalloidin for F-actin structures

  • Appropriate organelle markers for co-localization studies

• Live-cell imaging: For real-time translocation studies, express GFP-tagged RIPOR2 in appropriate cell lines and monitor movement using confocal microscopy with environmental controls (37°C, 5% CO₂) .

• Quantification: Measure the percentage of RIPOR2 signal in different subcellular compartments using fluorescence intensity ratios across at least 30 cells per condition .

This approach has successfully captured RIPOR2 translocation dynamics in response to aminoglycoside exposure in cochlear hair cells .

How can I effectively use RIPOR2 antibodies in multi-color immunofluorescence to study its role in immune cell polarization?

For multi-color immunofluorescence studies of RIPOR2 in immune cell polarization:

• Antibody panel selection:

  • Anti-RIPOR2 (rabbit polyclonal, unconjugated)

  • Anti-CD8 for T cells (mouse monoclonal)

  • Anti-RhoA for co-localization studies (goat polyclonal)

  • Appropriate actin/tubulin markers for cytoskeletal organization

• Sequential staining protocol:

  • Begin with RIPOR2 primary antibody (1:150) overnight at 4°C

  • Apply secondary antibody (Alexa-647 goat anti-rabbit)

  • Block with excess rabbit IgG before subsequent primary antibodies

  • Apply remaining primary antibodies followed by spectrally distinct secondary antibodies

  • Counterstain nuclei with DAPI

• Controls:

  • Single-color controls for spectral compensation

  • FMO (fluorescence minus one) controls to set proper gates

  • Isotype controls to verify specificity

• Imaging parameters:

  • Use confocal microscopy with sequential scanning

  • Maintain consistent exposure settings across experimental groups

  • Capture z-stacks (0.5 μm steps) to analyze entire cell volume

• Quantitative analysis:

  • Measure polarization index (ratio of RIPOR2 intensity at leading vs. trailing edge)

  • Calculate Pearson's correlation coefficient for co-localization with RhoA

  • Analyze cytoskeletal orientation relative to RIPOR2 distribution

This methodology enables comprehensive assessment of RIPOR2's spatial organization during immune cell polarization events.

How does RIPOR2 expression correlate with immune checkpoint inhibitor response in cancer research?

Studies have established significant correlations between RIPOR2 expression and immunotherapy response:

• Expression patterns: High RIPOR2 expression is associated with lower tumor mutation burden (TMB), higher ESTIMATEScores (reflecting immune and stromal cell presence in tumors), and elevated immune checkpoint (ICP) expression .

• Immune phenotype correlation: RIPOR2 expression levels correlate with tumor immune phenotypes:

  • High RIPOR2: "Inflamed" phenotype with greater immune cell infiltration

  • Low RIPOR2: "Desert" immune phenotype with minimal immune cell presence

• Immunotherapy response prediction: Patients with high RIPOR2 expression show significantly better responses to:

  • PD-1 inhibitor monotherapy (p < 0.05)

  • Combined PD-1/CTLA4 blockade (p < 0.01)

• Immune cell composition: High RIPOR2 expression correlates with:

  • Increased CD8+ T cell infiltration

  • Higher levels of activated CD4+ memory T cells

  • Enhanced B cell presence (confirmed via multiple computational methods: TIMER, XCELL, MCP-counter, quanTIseq, and EPIC)

• Checkpoint expression correlation: RIPOR2 expression positively correlates with multiple immune checkpoints including LAG3, TIGIT, CTSS, ICOS, and TIM3, providing a potential mechanistic explanation for immunotherapy responsiveness .

These findings suggest RIPOR2 could serve as a predictive biomarker for immunotherapy response, particularly in cervical cancer patients .

What methodological approaches are recommended for investigating RIPOR2's role in hearing loss using RIPOR2 antibodies?

For investigating RIPOR2's role in hearing loss:

• Tissue preparation techniques:

  • For cochlear sections: 4% PFA fixation followed by decalcification using EDTA (0.5M, pH 8.0) for 72 hours before paraffin embedding

  • For whole-mount preparations: Careful dissection of the organ of Corti followed by immediate fixation

• Antibody validation in knockout models:

  • Use tissues from Ripor2+/− and Ripor2−/− mice to confirm antibody specificity

  • Compare staining patterns between heterozygous and wild-type samples to assess dose-dependent expression

• Co-labeling strategy:

  • Combine anti-RIPOR2 antibody with:

    • Anti-parvalbumin (for hair cell identification)

    • Phalloidin (for F-actin/stereocilia visualization)

    • GFP antibodies (when using reporter constructs)

• Subcellular localization analysis:

  • High-resolution confocal microscopy (minimum 63x objective with 2x zoom)

  • Super-resolution techniques (STED or STORM) for precise localization at stereocilia base

  • Z-stack analysis with 0.25 μm steps through the entire hair cell

• Functional correlation studies:

  • Combine immunohistochemistry with auditory brainstem response (ABR) measurements

  • Correlate RIPOR2 localization changes with hearing thresholds after aminoglycoside exposure

  • Use calcium imaging alongside RIPOR2 immunostaining to link localization with mechanotransduction

This methodological approach has successfully demonstrated RIPOR2's critical role in stereocilia maintenance and aminoglycoside-induced ototoxicity .

How can RIPOR2 antibodies be utilized to investigate its role in genomic instability in cancer research?

To investigate RIPOR2's role in genomic instability:

• Combined immunohistochemistry and DNA damage marker analysis:

  • Perform dual staining of tumor sections with:

    • Anti-RIPOR2 antibody (1:20-1:200 dilution)

    • DNA damage markers (γH2AX, 53BP1, RAD51)

  • Quantify co-localization patterns and correlation with tumor stage

• Cell line model systems:

  • Establish RIPOR2-overexpressing cell lines (as done in SiHa and HeLa cells)

  • Combine with DNA damage induction (radiation, cisplatin)

  • Assess repair kinetics through time-course immunofluorescence analysis

• Protein interaction networks:

  • Use RIPOR2 antibodies for co-immunoprecipitation followed by mass spectrometry

  • Focus on interactions with DNA damage response (DDR) proteins

  • Validate key interactions (particularly with PARP1) through reciprocal IP and Western blotting

• Correlation with mutation signatures:

  • Combine RIPOR2 IHC scoring with whole-genome or exome sequencing data

  • Analyze relationships between RIPOR2 expression levels and specific mutation signatures

  • Correlate with microsatellite instability (MSI) status across tumor samples

• Functional readouts:

  • Measure DDR-related gene expression (FEN1, PARP1, PARP2, RAD51, ATM) in cells with varied RIPOR2 expression

  • Assess chromosomal aberrations using metaphase spread analysis

  • Quantify micronuclei formation as a marker of genomic instability

This comprehensive approach has revealed that low RIPOR2 expression correlates with increased genomic instability, higher tumor mutation burden, and differential expression of DDR-related genes in cervical cancer .

What are the most common issues when using RIPOR2 antibodies in Western blotting and how can they be resolved?

Common issues and solutions for RIPOR2 Western blotting:

Additionally, use 4-10% gradient gels for optimal separation, transfer at 30V overnight at 4°C for high molecular weight RIPOR2, and validate antibody specificity using RIPOR2 knockdown/knockout controls when possible .

How can I address inconsistent results in RIPOR2 immunohistochemistry staining of paraffin-embedded tissues?

To address inconsistent RIPOR2 IHC staining:

• Antigen retrieval optimization:

  • Test multiple methods systematically:

    • Heat-induced epitope retrieval using citrate buffer (pH 6.0)

    • EDTA buffer (pH 9.0)

    • Enzymatic retrieval using proteinase K

  • Optimize duration (10-30 minutes) and temperature (95-125°C)

• Fixation considerations:

  • Limit fixation time to 24-48 hours

  • For archival samples, increase antigen retrieval time

  • Consider dual antigen retrieval approach (heat followed by enzymatic)

• Antibody dilution series:

  • Test broad range (1:20 to 1:200) as recommended

  • Prepare fresh dilutions from concentrated stock

  • Incubate at 4°C overnight rather than room temperature

• Signal amplification systems:

  • Standard ABC method may provide insufficient signal

  • Try polymer-based detection systems

  • For low expression samples, implement tyramide signal amplification

• Validation controls:

  • Include known positive tissue (fetal myoblasts)

  • Use early-stage cervical cancer tissues as positive control

  • Implement on-slide positive and negative controls

• Counterstaining optimization:

  • Brief hematoxylin counterstaining (10-15 seconds)

  • Gentle bluing in lithium carbonate

  • Consider automated staining platforms for consistency

This systematic approach has successfully resolved inconsistent staining issues in RIPOR2 IHC studies of cervical cancer progression .

What strategies can overcome challenges in detecting endogenous RIPOR2 in primary immune cells using immunofluorescence?

For improved detection of endogenous RIPOR2 in primary immune cells:

• Sample preparation optimization:

  • For peripheral blood immune cells: Purify by magnetic separation before fixation

  • For tissue-resident immune cells: Minimize enzymatic digestion time

  • Use cytospin preparation (300g, 5 minutes) for adherence to slides

• Fixation and permeabilization testing matrix:

  Fixative	Permeabilization	Results
  4% PFA, 10 min	0.1% Triton X-100, 5 min	Preserves morphology but may reduce signal
  2% PFA, 5 min	0.5% Saponin, 10 min	Better for membrane-associated RIPOR2
  Methanol, -20°C, 10 min	No additional step	Best for cytoskeletal-associated RIPOR2
  1:1 Methanol:Acetone, -20°C, 5 min	No additional step	Balanced approach for most immune cells

• Signal amplification techniques:

  • Implement tyramide signal amplification (TSA) system

  • Use high-sensitivity fluorophores (Alexa Fluor Plus series)

  • Consider quantum dots for low abundance detection

• Background reduction strategies:

  • Pre-adsorb secondary antibodies with cell lysates

  • Include 10% serum from host species of secondary antibody

  • Add 0.1-0.3M glycine to reduce aldehyde-induced autofluorescence

• Advanced imaging approaches:

  • Deconvolution microscopy for improved signal-to-noise ratio

  • Airyscan or structured illumination for super-resolution capabilities

  • Image stacks with constrained iterative deconvolution

These strategies have enabled successful detection of endogenous RIPOR2 in primary T cells and neutrophils, revealing its dynamic localization during immune cell polarization .

How should researchers interpret contradictory RIPOR2 expression data between antibody-based methods and RNA-sequencing results?

When facing contradictions between antibody-based and RNA-seq RIPOR2 data:

• Evaluate post-transcriptional regulation:

  • RIPOR2 may undergo significant post-transcriptional regulation

  • Calculate protein-to-mRNA ratios across samples to identify patterns

  • Investigate miRNA regulation using correlation analysis with known RIPOR2-targeting miRNAs

• Assess protein stability and turnover:

  • Measure RIPOR2 half-life using cycloheximide chase assays

  • Compare stability across different experimental conditions

  • Investigate proteasomal degradation using inhibitors like MG132

• Consider isoform-specific detection limitations:

  • RNA-seq may detect all transcript variants

  • Antibodies may recognize specific epitopes present in only certain isoforms

  • Perform isoform-specific RT-PCR for validation

• Technical validation strategies:

  • Use multiple antibodies targeting different RIPOR2 epitopes

  • Implement orthogonal protein detection methods (mass spectrometry)

  • Design experiments with RIPOR2 overexpression and knockdown controls

• Biological context consideration:

  • Examine cell-type specific differences in post-transcriptional regulation

  • Assess subcellular localization which may affect extraction efficiency

  • Consider activation-dependent translocation affecting antibody accessibility

This comprehensive approach revealed that in cervical cancer, discrepancies between RIPOR2 protein and mRNA levels were attributed to differential protein stability and subcellular translocation rather than technical limitations of detection methods .

What are the best experimental designs to investigate RIPOR2's dynamic interactions with the cytoskeleton using antibody-based approaches?

To investigate RIPOR2-cytoskeleton interactions:

• Live-cell imaging setup:

  • Express fluorescently-tagged RIPOR2 constructs (confirm functionality)

  • Co-express markers for cytoskeletal components (LifeAct for F-actin)

  • Implement spinning disk confocal microscopy with environmental control

  • Capture images every 5-10 seconds during cell polarization events

• Proximity ligation assay (PLA) design:

  • Combine anti-RIPOR2 antibody with antibodies against:

    • Actin

    • Myosin

    • RhoA

    • Other cytoskeletal regulatory proteins

  • Quantify interaction signals at different cellular locations and timepoints

  • Include competitive inhibition controls

• Subcellular fractionation approach:

  • Separate cytosolic, membrane, cytoskeletal, and nuclear fractions

  • Analyze RIPOR2 distribution across fractions via Western blotting

  • Track changes in distribution after cytoskeletal-disrupting drugs

  • Include markers for each fraction (α-tubulin, Na+/K+-ATPase, lamin)

• Structured drug perturbation experiments:

  Cytoskeletal Drug	Concentration	Pretreatment Time	Expected Effect on RIPOR2
  Latrunculin B	1-5 μM	30 min	Disrupts actin-RIPOR2 interactions
  Jasplakinolide	0.5-1 μM	30 min	Stabilizes actin-RIPOR2 interactions
  Y-27632 (ROCK inhibitor)	10-20 μM	2 hours	Blocks RhoA-mediated effects on RIPOR2
  Blebbistatin	10-50 μM	1 hour	Reveals myosin-dependent RIPOR2 localization

• FRAP (Fluorescence Recovery After Photobleaching) analysis:

  • Express GFP-RIPOR2 in appropriate cell models

  • Photobleach regions of interest at cytoskeletal structures

  • Measure recovery kinetics to determine mobile/immobile fractions

  • Compare between different cytoskeletal regions and after drug treatments

This multi-modal approach has revealed RIPOR2's dynamic interactions with actin during T cell polarization and stereocilia maintenance in hair cells .

What controls and validation steps are essential when using RIPOR2 antibodies in studies involving genetic manipulation of RIPOR2 expression?

When using RIPOR2 antibodies with genetic manipulation:

• Essential expression vector controls:

  • Empty vector controls for overexpression studies

  • Non-targeting scrambled controls for knockdown experiments

  • Rescue experiments with siRNA-resistant constructs

  • Dose-response studies with varying levels of overexpression

• Antibody validation in genetically modified systems:

  • Test antibody in RIPOR2 knockout/knockdown systems to confirm specificity

  • Validate detection of overexpressed tagged constructs

  • Compare multiple antibodies targeting different epitopes

  • Perform epitope mapping if unexpected results occur

• Quantification standards:

  • Establish standard curves using recombinant RIPOR2 protein

  • Normalize to appropriate housekeeping proteins (GAPDH, β-actin)

  • Implement density-based quantification with background subtraction

  • Use digital PCR for absolute copy number determination of transcripts

• Functional validation:

  • Confirm phenotypic changes match expected outcomes (migration, proliferation)

  • Verify effects on established RIPOR2 downstream targets (RhoA activity)

  • Assess restoration of function with rescue constructs

  • Implement multiple independent knockdown/overexpression methods

• Off-target effect assessment:

  • Monitor related family members (RIPOR1, RIPOR3) for compensatory changes

  • Check for effects on known interacting partners

  • Perform RNA-seq to detect broader transcriptional changes

  • Use CRISPR/Cas9 with multiple guide RNAs for validation

These comprehensive validation steps were crucial in establishing RIPOR2's role as an anti-tumor factor in cervical cancer through multiple experimental approaches including EdU assays, colony formation assays, and transwell migration assays .

How can RIPOR2 antibodies be integrated into multiplexed proteomic approaches to study cancer immune microenvironments?

For multiplexed proteomic studies of RIPOR2 in cancer immune microenvironments:

• Multiplex immunofluorescence panel design:

  • Basic panel: Anti-RIPOR2 + CD8 + PD-1 + tumor marker

  • Advanced panel: Add CD4, FOXP3, CD68, and tumor-specific markers

  • Ultra-high parameter panel: Implement Vectra/Polaris systems with 7+ markers

  • Consider compatibility of primary antibody species to minimize crosstalk

• Sequential multiplexed immunohistochemistry:

  • Apply antibody, detect, image, then strip and repeat

  • Validate stripping efficiency between rounds

  • Include registration markers for accurate image overlay

  • Enable detection of 10+ markers on a single section

• Mass cytometry (CyTOF) integration:

  • Metal-conjugated anti-RIPOR2 antibodies (typically 165Ho or 166Er)

  • Panel design including 30+ immune markers

  • Focus on markers with established RIPOR2 correlations (LAG3, TIGIT, CTSS, etc.)

  • Implement viSNE or UMAP dimensionality reduction for visualization

• Digital spatial profiling:

  • ROI selection based on RIPOR2 expression patterns

  • Apply DSP technology with UV-cleavable oligo-conjugated antibodies

  • Quantify spatial relationships between RIPOR2+ cells and immune populations

  • Correlate with genomic instability markers

• Single-cell proteogenomic correlation:

  • CITE-seq approaches combining RIPOR2 antibody with transcriptomics

  • Correlate protein vs. mRNA at single-cell resolution

  • Trace lineage relationships in RIPOR2+ cells within tumor microenvironment

  • Identify cellular neighborhoods with distinct RIPOR2 expression patterns

This multiplexed approach revealed that tumors with high RIPOR2 expression harbor significantly higher CD8+ T cell infiltration and exhibit an "inflamed" phenotype that correlates with better immunotherapy response .

What are the methodological considerations for investigating the relationship between RIPOR2 and DNA damage response proteins in genomic instability research?

For investigating RIPOR2-DDR protein relationships:

• Co-immunoprecipitation optimization:

  • Crosslinking approach: DSP (1 mM, 30 min) preserves transient interactions

  • Lysis buffer: Include 1 mM MgCl₂, 5 mM NaF, 1 mM Na₃VO₄ to maintain phosphorylation status

  • Pre-clearing: 1 hour with protein A/G beads to reduce background

  • Antibody ratio: 5 μg anti-RIPOR2 antibody per 500 μg protein lysate

• DNA damage induction panel:

  DNA Damage Type	Inducing Agent	Concentration/Dose	Primary DDR Pathways Activated
  Double-strand breaks	Ionizing radiation	2-10 Gy	NHEJ, HR (ATM-dependent)
  Replication stress	Hydroxyurea	0.5-2 mM, 6-24h	ATR-CHK1 pathway
  Interstrand crosslinks	Cisplatin	10-50 μM, 4-24h	FA pathway, NER, HR
  Base damage	H₂O₂	100-500 μM, 0.5-2h	BER (PARP1-dependent)

• Proximity ligation assay (PLA) optimization:

  • Test multiple antibody pairs (RIPOR2 with PARP1, ATM, γH2AX, etc.)

  • Include technical controls (single primary antibodies)

  • Optimize primary antibody concentration (typically 1:100-1:500)

  • Quantify PLA signals per nucleus across >100 cells per condition

• Chromatin fractionation approach:

  • Separate soluble nuclear proteins from chromatin-bound fractions

  • Assess RIPOR2 recruitment to chromatin after DNA damage

  • Include positive controls (PARP1, γH2AX) that localize to chromatin after damage

  • Monitor time-course of recruitment/resolution

• Functional DNA repair assays:

  • HR reporter assay in RIPOR2-manipulated cells

  • NHEJ reporter assay in RIPOR2-manipulated cells

  • Comet assay to measure DNA damage resolution kinetics

  • RAD51 and 53BP1 foci formation and resolution

This comprehensive approach revealed that RIPOR2 expression inversely correlates with genomic instability markers and affects the expression of multiple DDR genes, with a particularly strong relationship with PARP1 expression in cervical cancer cells .

What approaches can be used to investigate RIPOR2's role in aminoglycoside-induced ototoxicity using RIPOR2 antibodies?

For investigating RIPOR2 in aminoglycoside-induced ototoxicity:

• Ex vivo cochlear explant model:

  • Harvest cochlear explants from P4 mice

  • Treat with 1 mM gentamicin for time intervals (2.5-120 minutes)

  • Process for immunofluorescence with anti-RIPOR2 (1:150) and phalloidin

  • Analyze changes in subcellular localization using high-resolution confocal microscopy

• Protein-drug interaction analysis:

  • GEN-conjugated bead pull-down assays with recombinant RIPOR2

  • Competition assays with soluble aminoglycosides

  • Direct binding assays with purified proteins

  • Correlation of binding affinity with ototoxicity potential of different aminoglycosides

• In vivo model development:

  • Utilize Ripor2+/− mice for dose-response studies

  • Administer kanamycin (800-1200 mg/kg)

  • Assess hearing function via auditory brainstem response (ABR)

  • Correlate cochlear histopathology with RIPOR2 expression levels and localization

• Mechanistic pathway analysis:

  • Co-immunoprecipitation of RIPOR2 with autophagy proteins

  • Western blot assessment of autophagy markers (LC3, p62)

  • Proximity ligation assays between RIPOR2 and GABARAP/GABARAPL1

  • Functional autophagy assays in hair cells with varied RIPOR2 expression

• Translational intervention approach:

  • Test protective compounds that stabilize RIPOR2 localization

  • Assess autophagy modulators as protective agents

  • Implement gene therapy approaches to modify RIPOR2 expression

  • Evaluate otoprotective potential through functional and histological measures