RTP3 Antibody

What is RTP3 protein and why is it significant in research settings?

RTP3 (Receptor Transporter Protein 3) is a membrane-localized protein with a length of 232 amino acid residues and a molecular weight of approximately 27 kDa in humans . It belongs to the TMEM7 protein family and plays a crucial role in promoting functional cell surface expression of bitter taste receptors, specifically TAS2R16 and TAS2R43 . The significance of RTP3 in research stems from its high expression in adult liver tissue and its conserved presence across several mammalian species including mouse, rat, bovine, and chimpanzee . Understanding RTP3's function has implications for taste perception research, membrane protein trafficking, and potentially liver-specific cellular functions.

What epitope targets are available for RTP3 antibodies and how does epitope selection affect experimental outcomes?

RTP3 antibodies target various epitope regions of the protein, with commercially available options covering different amino acid sequences including AA 1-211, AA 21-120, and AA 81-180 . The choice of epitope significantly impacts experimental outcomes because:

• Accessibility: Membrane-embedded regions may be less accessible than extracellular domains

• Conservation: Epitopes with higher evolutionary conservation allow cross-species applications

• Post-translational modifications: Modifications near certain epitopes may block antibody binding

• Functional domains: Antibodies targeting functional domains may inhibit protein activity

For optimal results in functional studies, researchers should select antibodies targeting the AA 21-120 region, which includes important functional domains . For protein expression studies, antibodies against the AA 1-211 region provide broader coverage of the protein . The AA 81-180 region contains the sequence "RGQVKMRVFT QRCKKCPQPL FEDPEFTQEN ISRILKNLVF RILKKCYRGR FQLIEEVPMI KDISLEGPHN SDNCEACLQG FCAGPIQVTS LPPSQTPRVH" which includes several important functional motifs .

How do various host animals influence the properties of anti-RTP3 antibodies?

The choice of host animal in which anti-RTP3 antibodies are raised significantly influences antibody characteristics and experimental applications. Based on available information:

• Rabbit-derived polyclonal RTP3 antibodies demonstrate broad applicability across multiple techniques including Western Blot, ELISA, immunohistochemistry, and flow cytometry . These antibodies typically offer high sensitivity due to their recognition of multiple epitopes on the RTP3 protein.

• Mouse-derived polyclonal RTP3 antibodies show more limited application range, primarily validated for Western Blot and ELISA techniques . These antibodies may offer advantages when performing multi-color immunostaining alongside rabbit-derived antibodies against other targets.

The host species selection impacts:

• Background staining in tissues of the same species (increased background when using mouse antibodies on mouse tissues)

• Secondary antibody compatibility

• Recognition of post-translational modifications (which can vary based on the host's immune response)

• Cross-reactivity profile (rabbit polyclonals generally show broader cross-reactivity across species)

How should Western blotting protocols be optimized for RTP3 antibody detection?

Optimizing Western blotting protocols for RTP3 antibody detection requires careful consideration of several technical parameters:

• Sample preparation:

  • Use RIPA buffer with protease inhibitors for membrane protein extraction

  • Heat samples at 70°C (not 95°C) for 10 minutes to prevent membrane protein aggregation

  • Use fresh samples when possible as RTP3 can degrade during freeze-thaw cycles

• Gel electrophoresis:

  • Use 10-12% polyacrylamide gels to properly resolve the 27 kDa RTP3 protein

  • Load 20-30 μg of total protein per lane for liver samples (where RTP3 is highly expressed)

  • Include positive controls from liver tissue extracts

• Transfer conditions:

  • Use PVDF membranes (0.45 μm pore size) for optimal protein binding

  • Transfer at 100V for 1 hour in 10% methanol-containing transfer buffer

  • Verify transfer efficiency with Ponceau S staining

• Antibody incubation:

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

  • Dilute primary RTP3 antibodies 1:500 to 1:1000 depending on specific product

  • Incubate with primary antibody overnight at 4°C

  • Use appropriate species-specific HRP-conjugated secondary antibodies at 1:5000 dilution

• Detection:

  • Use enhanced chemiluminescence (ECL) detection reagents

  • Expect a band at approximately 27 kDa for human RTP3

This methodology has been validated with both rabbit-derived and mouse-derived anti-RTP3 antibodies across multiple studies .

What are the critical validation steps for ensuring RTP3 antibody specificity?

Ensuring antibody specificity is crucial for generating reliable research data. For RTP3 antibodies, implement these validation steps:

• Positive and negative controls:

  • Use liver tissue (high RTP3 expression) as a positive control

  • Compare with tissues known to have low RTP3 expression

  • Include RTP3-knockout or siRNA-treated samples as negative controls

• Peptide competition assay:

  • Pre-incubate the antibody with excess immunizing peptide (e.g., the specific AA sequence used to generate the antibody)

  • Run parallel Western blots or IHC with blocked and unblocked antibody

  • Specific binding should be eliminated in the peptide-blocked sample

• Cross-reactivity assessment:

  • Test the antibody against recombinant RTP3 and closely related family members

  • Verify signal reduction in samples with reduced RTP3 expression

  • Check antibody reactivity in multiple species if cross-species reactivity is claimed

• Orthogonal method validation:

  • Compare results from different antibody clones targeting distinct RTP3 epitopes

  • Validate findings using nucleic acid-based methods (RT-PCR, RNA-seq)

  • Consider using tagged RTP3 constructs for expression with subsequent detection by tag-specific antibodies

• Mass spectrometry confirmation:

  • Immunoprecipitate RTP3 using the antibody

  • Confirm the identity of the precipitated protein by mass spectrometry

  • This provides definitive evidence of antibody specificity

These validation steps significantly enhance confidence in experimental results and minimize the risk of antibody-based artifacts .

What are the optimal conditions for immunohistochemical detection of RTP3 in tissue sections?

Optimizing immunohistochemical (IHC) detection of RTP3 in tissue sections requires attention to several critical parameters:

• Tissue preparation:

  • Fix tissues in 10% neutral-buffered formalin for 24 hours

  • Process and embed in paraffin following standard protocols

  • Cut sections at 4-5 μm thickness

• Antigen retrieval (critical for RTP3 detection):

  • Heat-induced epitope retrieval in citrate buffer (pH 6.0) for 20 minutes

  • Alternative: EDTA buffer (pH 9.0) if citrate buffer yields insufficient signal

  • Allow slides to cool slowly to room temperature

• Blocking and antibody incubation:

  • Block endogenous peroxidase with 3% hydrogen peroxide for 10 minutes

  • Apply protein block (5% normal serum) for 30 minutes

  • Incubate with primary anti-RTP3 antibody at 1:100-1:200 dilution overnight at 4°C

  • Use antibodies validated for IHC-p (paraffin sections), particularly those targeting the AA 21-120 region

• Detection system:

  • Use biotin-free polymer detection systems to minimize background

  • Develop with DAB chromogen for 5-10 minutes (monitor under microscope)

  • Counterstain with hematoxylin for 1-2 minutes

• Controls and interpretation:

  • Include liver tissue as positive control

  • Use isotype control antibodies as negative controls

  • Expect membranous staining pattern in positive cells

  • Evaluate both staining intensity and percentage of positive cells

This protocol has been optimized based on the properties of available RTP3 antibodies and the membrane localization of the target protein .

How can RTP3 antibodies be utilized in co-immunoprecipitation studies to identify interacting proteins?

Co-immunoprecipitation (Co-IP) with RTP3 antibodies provides valuable insights into protein-protein interactions. The following methodological approach is recommended:

• Cell/tissue preparation:

  • Harvest cells or tissues expressing RTP3 (liver tissue or appropriate cell lines)

  • Lyse in gentle lysis buffer (1% NP-40, 150 mM NaCl, 50 mM Tris pH 7.4) with protease inhibitors

  • Clear lysates by centrifugation at 14,000 g for 15 minutes at 4°C

• Antibody selection and coupling:

  • Select RTP3 antibodies validated for immunoprecipitation

  • The antibody targeting AA 1-211 shows superior performance in IP applications

  • Pre-couple 2-5 μg antibody to Protein A/G magnetic beads for 1 hour at room temperature

• Immunoprecipitation:

  • Incubate cleared lysate with antibody-coupled beads overnight at 4°C with gentle rotation

  • Wash beads 4-5 times with cold lysis buffer containing reduced detergent (0.1% NP-40)

  • Elute bound proteins with either gentle elution buffer or by boiling in SDS sample buffer

• Analysis of interacting proteins:

  • Analyze by Western blot to detect known or suspected interacting proteins

  • For unbiased discovery, analyze by mass spectrometry

  • Focus on membrane proteins, particularly bitter taste receptors (TAS2R16, TAS2R43)

• Validation of interactions:

  • Confirm interactions by reverse Co-IP using antibodies against identified partners

  • Validate with in vitro binding assays using recombinant proteins

  • Consider proximity ligation assays to confirm interactions in intact cells

This approach enables researchers to map the RTP3 interactome, particularly focusing on its role in trafficking bitter taste receptors to the cell surface .

How can flow cytometry be optimized for detecting RTP3 expression in heterogeneous cell populations?

Optimizing flow cytometry for RTP3 detection requires specialized procedures due to its membrane localization:

• Sample preparation:

  • Harvest cells gently using non-enzymatic cell dissociation solution

  • Fix with 2% paraformaldehyde for 15 minutes at room temperature

  • Permeabilize with 0.1% saponin (not Triton X-100) to preserve membrane integrity

• Antibody selection and staining:

  • Use fluorochrome-conjugated anti-RTP3 antibodies when available (such as FITC-conjugated)

  • Alternatively, use primary anti-RTP3 antibodies validated for flow cytometry followed by fluorophore-conjugated secondary antibodies

  • Antibodies targeting the AA 21-120 region show optimal results for flow cytometry

  • Incubate with antibody in buffer containing 0.1% saponin for 45-60 minutes at 4°C

• Protocol optimization:

  • Titrate antibody concentration to determine optimal signal-to-noise ratio

  • Include appropriate isotype controls matched to primary antibody

  • Perform compensation controls when multiplexing with other markers

  • Use viability dye to exclude dead cells from analysis

• Data acquisition and analysis:

  • Collect at least 10,000 events per sample

  • Gate on single cells using FSC-A vs. FSC-H

  • Analyze RTP3 expression relative to known markers of target cell populations

  • Consider co-staining for bitter taste receptors to analyze co-expression patterns

• Validation strategies:

  • Include positive controls (liver-derived cells) and negative controls

  • Compare results with Western blot quantification

  • Verify specificity with competitive blocking using immunizing peptide

This approach allows quantitative assessment of RTP3 expression across different cell types and experimental conditions .

What experimental approaches can be used to study RTP3 subcellular localization and trafficking?

Investigating RTP3 subcellular localization and trafficking requires multi-modal approaches:

• Immunofluorescence microscopy:

  • Fix cells with 4% paraformaldehyde for 15 minutes

  • Permeabilize with 0.1% saponin to preserve membrane structures

  • Use anti-RTP3 antibodies at 1:100-1:200 dilution

  • Co-stain with markers for specific cellular compartments:

    • Plasma membrane (Na+/K+-ATPase)

    • Endoplasmic reticulum (calnexin)

    • Golgi apparatus (GM130)

    • Endosomes (Rab5, Rab7)

  • Analyze using confocal microscopy for precise co-localization assessment

• Live-cell imaging with fluorescent protein fusions:

  • Create RTP3-GFP or RTP3-mCherry fusion constructs

  • Transfect cells with minimal expression to avoid overexpression artifacts

  • Perform time-lapse imaging to track RTP3 trafficking

  • Use photoactivatable or photoconvertible tags for pulse-chase analysis

• Biochemical fractionation:

  • Separate cellular compartments using differential centrifugation

  • Prepare membrane, cytosolic, and nuclear fractions

  • Analyze RTP3 distribution by Western blotting using specific anti-RTP3 antibodies

  • Include markers for each fraction to confirm separation quality

• Proximity labeling approaches:

  • Create BioID or APEX2 fusions with RTP3

  • Identify proximal proteins in different cellular compartments

  • Map the spatial organization of RTP3 in the cell

• Super-resolution microscopy:

  • Apply STORM or PALM imaging using appropriately labeled secondary antibodies

  • Achieve nanometer-scale resolution of RTP3 distribution

  • Quantify clustering and co-localization with interacting proteins

These methodologies provide complementary data on RTP3's dynamic localization, particularly in relation to its role in trafficking bitter taste receptors to the cell surface .

What are common causes of false positive or false negative results when using RTP3 antibodies?

Understanding potential artifacts in RTP3 antibody-based experiments is critical for accurate data interpretation:

• Common causes of false positive results:

  • Cross-reactivity with structurally similar proteins, particularly other TMEM family members

  • Non-specific binding to highly expressed liver proteins

  • Inappropriate secondary antibody selection causing background signal

  • Insufficient blocking leading to hydrophobic interactions with membrane fractions

  • Over-development of immunohistochemistry or Western blot signals

• Common causes of false negative results:

  • Epitope masking due to protein-protein interactions or post-translational modifications

  • Inadequate sample preparation failing to expose membrane-embedded epitopes

  • Insufficient antigen retrieval in fixed tissues

  • Protein degradation during sample preparation

  • Sub-optimal antibody concentration or incubation conditions

• Mitigation strategies:

  • Always include positive and negative controls

  • Validate results with at least two antibodies targeting different epitopes

  • Perform peptide competition assays to confirm specificity

  • Use genetic approaches (knockdown/knockout) to validate antibody specificity

  • Consider the native expression level of RTP3 in your experimental system

• Technical considerations:

  • For Western blot: ensure complete protein transfer and appropriate blocking

  • For IHC: optimize antigen retrieval and minimize background with appropriate blockers

  • For flow cytometry: careful gating and compensation to avoid autofluorescence artifacts

These considerations help researchers distinguish genuine RTP3 signals from technical artifacts .

How should researchers interpret discrepancies in RTP3 detection between different antibody-based methods?

When faced with discrepancies between different antibody-based detection methods for RTP3, employ this systematic approach to interpretation:

• Method-specific considerations:

  • Western blot detects denatured protein and reveals molecular weight information

  • IHC preserves tissue architecture but may have reduced epitope accessibility

  • Flow cytometry provides quantitative single-cell data but requires cell dissociation

  • IP methods depend on native protein conformation and accessibility in solution

• Epitope-dependent factors:

  • Different epitopes may be differentially accessible in various applications

  • Antibodies targeting AA 21-120 perform better in IHC and flow cytometry applications

  • Antibodies targeting AA 1-211 may be superior for Western blot applications

  • Post-translational modifications may block specific epitopes in a context-dependent manner

• Hierarchical resolution approach:

  • Determine which method provides higher specificity through controls

  • Consider orthogonal methods (mRNA analysis, tagged protein expression)

  • Evaluate which method aligns with known biology of RTP3

  • Assess technical quality and reproducibility of each method

• Integration strategy:

  • Create a consensus view based on multiple methods

  • Weight results based on validated controls for each method

  • Document methodological variables that may explain discrepancies

  • Consider biological variables (splice variants, protein modifications)

This approach enables researchers to resolve apparent contradictions and develop a more accurate understanding of RTP3 biology across experimental systems .

What quantification methods are most appropriate for RTP3 antibody signals in different applications?

Selecting appropriate quantification methods for RTP3 antibody signals is critical for generating reliable and reproducible data:

• Western blot quantification:

  • Use densitometry with background subtraction

  • Normalize to appropriate loading controls (β-actin, GAPDH)

  • For membrane proteins like RTP3, consider normalizing to Na+/K+-ATPase or cadherin

  • Apply linear range detection methods (avoid saturated signals)

  • Present data as fold change relative to controls

• Immunohistochemistry quantification:

  • Use H-score system (intensity × percentage of positive cells)

  • Alternative: Automated image analysis with tissue segmentation

  • Score membrane staining separately from cytoplasmic signals

  • Blind scoring by multiple observers to reduce bias

  • Include scale bars and representative images at multiple magnifications

• Flow cytometry quantification:

  • Report median fluorescence intensity (MFI) rather than mean

  • Calculate signal-to-noise ratio relative to isotype controls

  • Use molecules of equivalent soluble fluorochrome (MESF) for absolute quantification

  • Apply proper compensation when multiplexing

  • Present percentage of positive cells based on objective gating strategies

• Immunoprecipitation quantification:

  • Normalize to input material

  • Compare to IgG control pull-downs

  • Consider semi-quantitative ranking of interaction strengths

  • Include loading controls for input samples

• Statistical analysis:

  • Apply appropriate statistical tests based on data distribution

  • Report biological and technical replicates separately

  • Consider power analysis to determine adequate sample sizes

  • Use non-parametric tests when appropriate for IHC scoring

These quantification strategies enhance the rigor and reproducibility of RTP3 antibody-based research .

What criteria should guide selection between monoclonal and polyclonal RTP3 antibodies for specific applications?

The decision between monoclonal and polyclonal RTP3 antibodies should be guided by application-specific requirements:

• Polyclonal RTP3 antibodies advantages:

  • Higher sensitivity due to recognition of multiple epitopes

  • Greater tolerance to minor protein denaturation or modifications

  • Better performance in applications requiring high detection sensitivity

  • Currently more widely validated across multiple applications

  • Available from rabbit and mouse host species with various conjugations

• Monoclonal RTP3 antibodies advantages:

  • Superior reproducibility between experiments and lots

  • Higher specificity for particular epitopes

  • Reduced batch-to-batch variation

  • Better performance in quantitative applications requiring precise standardization

  • Lower background in some applications

• Application-specific recommendations:

  • For Western blot: Both types work well, with polyclonals providing higher sensitivity

  • For IHC: Polyclonal antibodies often provide stronger signal in paraffin sections

  • For flow cytometry: Monoclonals may provide more consistent results

  • For quantitative studies: Monoclonals offer better standardization

  • For detecting modified forms: Polyclonals that recognize multiple epitopes

• Selection matrix for RTP3 antibodies:

This guidance helps researchers select the most appropriate antibody format for their specific experimental goals .

How do different epitope-targeting strategies affect RTP3 antibody performance in various experimental systems?

The epitope-targeting strategy significantly influences RTP3 antibody performance across experimental systems:

• N-terminal region antibodies (AA 1-211):

  • Advantages: Detect full-length protein, useful for expression studies

  • Performance: Superior for immunoprecipitation applications

  • Limitations: May miss truncated variants or be affected by N-terminal modifications

  • Best applications: Western blot, IP, general protein expression studies

• Middle region antibodies (AA 21-120):

  • Advantages: Target functionally important domains, balanced accessibility

  • Performance: Excellent for multiple applications including IHC and flow cytometry

  • Limitations: May be affected by protein-protein interactions in this region

  • Best applications: Flow cytometry, IHC, functional studies

• Internal region antibodies (AA 81-180):

  • Advantages: Target conserved functional domains, including zinc finger motifs

  • Performance: High specificity in Western blot applications

  • Limitations: Reduced accessibility in native conformation

  • Best applications: Western blot, ELISA, specific domain studies

• Experimental system considerations:

  • Cell lines: Middle region antibodies perform well in most cultured cell systems

  • Tissues: Antibodies targeting AA 21-120 show optimal IHC performance in FFPE tissues

  • Recombinant systems: N-terminal antibodies useful for tagged-protein detection

  • Species cross-reactivity: Internal epitopes show higher conservation across species

• Functional domain targeting:

  • Zinc finger domains: Use AA 81-180 antibodies to study DNA binding functions

  • Membrane-associated regions: AA 21-120 antibodies for trafficking studies

  • Protein-protein interaction domains: Consider the impact of antibody binding on complex formation

This knowledge enables researchers to strategically select antibodies based on their specific experimental questions and systems .

What are the critical experimental controls needed when using RTP3 antibodies for functional studies?

Implementing robust controls is essential for generating reliable data with RTP3 antibodies in functional studies:

• Antibody specificity controls:

  • Peptide competition/blocking: Pre-incubate antibody with immunizing peptide

  • Genetic validation: Use RTP3 knockout/knockdown samples

  • Recombinant protein: Test antibody against purified RTP3 protein

  • Overexpression: Compare endogenous signal with overexpressed RTP3

• Technical controls:

  • Isotype control: Same species and isotype as primary antibody

  • Secondary-only control: Omit primary antibody to assess non-specific binding

  • Loading/processing controls: Ensure equal sample preparation across conditions

  • Positive tissue control: Include liver samples (high RTP3 expression)

• Functional study-specific controls:

  • For trafficking studies:

    • Compare wild-type RTP3 with trafficking-deficient mutants

    • Use known trafficking inhibitors as positive controls

    • Include co-expression with established RTP3 partners (TAS2R16, TAS2R43)

  • For protein-protein interaction studies:

    • Perform reciprocal co-immunoprecipitations

    • Include non-interacting protein controls

    • Verify interactions with multiple antibodies targeting different epitopes

• Quantitative controls:

  • Standard curves with recombinant protein (for quantitative applications)

  • Internal reference samples across experiments for normalization

  • Biological replicates from independent sources

  • Technical replicates to assess method reproducibility

• Documentation requirements:

  • Record complete antibody information (supplier, catalog number, lot number)

  • Document detailed experimental conditions

  • Maintain original unprocessed data alongside analyzed results

  • Report all controls in publications and presentations

What emerging applications for RTP3 antibodies are driving current research innovations?

The field of RTP3 antibody applications continues to evolve with several emerging areas of research significance:

• Single-cell analysis applications:

  • Integration of RTP3 antibodies in mass cytometry (CyTOF) panels for deep phenotyping

  • Single-cell Western blotting to examine RTP3 expression heterogeneity

  • Spatial proteomics using antibody-based detection methods to map RTP3 distribution within tissue microenvironments

  • Combined protein-transcript analysis at single-cell resolution using antibodies with in situ hybridization

• High-throughput screening applications:

  • Development of RTP3 antibody-based biosensors for real-time trafficking studies

  • Incorporation into automated immunoassay platforms for biomarker quantification

  • Application in cell-based screens to identify modulators of bitter taste receptor trafficking

• Advanced imaging techniques:

  • Super-resolution microscopy with specialized secondary antibody conjugates

  • Expansion microscopy for nanoscale visualization of RTP3 distribution

  • Cryo-electron tomography using antibody-gold labeling for 3D ultrastructural analysis

  • Intravital imaging using minimally invasive antibody-based probes

• Therapeutic and diagnostic development:

  • Internalization studies to assess RTP3 as a potential drug delivery target

  • Development of conformation-specific antibodies to distinguish functional states

  • Exploration of RTP3's role in liver pathophysiology using specific antibodies

These emerging applications represent cutting-edge directions in RTP3 research, building upon fundamental knowledge of its structure and function while leveraging technological advances in antibody-based detection methods .

How can researchers evaluate and report RTP3 antibody validation for publication purposes?

When preparing RTP3 antibody data for publication, researchers should follow these comprehensive validation and reporting guidelines:

• Essential validation experiments to perform:

  • Specificity testing using positive and negative controls

  • Demonstration of expected molecular weight by Western blot

  • Peptide competition assays showing signal reduction

  • Correlation with mRNA expression or orthogonal protein detection methods

  • Application-specific controls (as detailed in section 5.3)

• Complete antibody documentation:

  • Commercial source, catalog number, and lot number

  • Clone identifier for monoclonals or immunogen details for polyclonals

  • Host species and antibody isotype

  • Exact epitope or immunogen sequence

  • RRID (Research Resource Identifier) when available

• Detailed methodology reporting:

  • Antibody concentration/dilution used

  • Incubation conditions (time, temperature, buffer composition)

  • Detection methods (secondary antibodies, detection chemistry)

  • Image acquisition parameters (exposure, gain settings)

  • Quantification methods with statistical approaches

• Transparent data presentation:

  • Include full unprocessed blot images with molecular weight markers

  • Show representative images alongside quantification

  • Present both positive and negative results

  • Include all relevant controls in figures or supplements

  • Declare any antibody validation limitations

• Adherence to community guidelines:

  • Follow journal-specific antibody reporting requirements

  • Consider the International Working Group for Antibody Validation (IWGAV) guidelines

  • Address the "Five Pillars" of antibody validation when applicable