RPTOR Antibody

What is RPTOR and why is it important in cellular signaling pathways?

RPTOR (Regulatory Associated Protein of mTOR, Complex 1) is a 150 kDa component of the cytosolic mammalian target of Rapamycin complex 1 (mTORC1), which also contains mTOR and GBL proteins. It functions as a scaffold protein whose binding by TOR substrates is necessary for effective TOR-catalyzed phosphorylation. These substrates include the ribosomal protein S6 kinase (RP S6K) and the eukaryotic initiation factor 4E binding protein 4EBP1, which are essential for cell growth and proliferation in response to nutrient and mitogen levels .

The interaction of RPTOR with mTOR is stabilized under conditions of nutrient deprivation and energy stress, leading to inhibition of mTOR and cell cycle arrest. RPTOR contains multiple Ser and Thr residues whose phosphorylation regulates the activation status of mTOR . The protein is critical for the response of skeletal muscle and adipose tissue to insulin and contains specific structural domains: three RNC blocks (aa 48-511), three HEAT repeats (aa 550-667), and seven C-terminal WD40 domains (aa 1020-1335) .

How should I select the appropriate RPTOR antibody for my specific experimental application?

When selecting an RPTOR antibody for research, consider these critical factors:

• Application compatibility: Verify the antibody has been validated for your specific technique (Western blot, IHC, ICC, IF, flow cytometry). Different applications require antibodies with distinct properties. For example, some antibodies work well in Western blot at 2-4 μg/mL but require higher concentrations (10 μg/mL) for immunocytochemistry or immunofluorescence .

• Species reactivity: Ensure the antibody recognizes RPTOR in your experimental species. Many RPTOR antibodies show reactivity to human and mouse proteins, with some also recognizing rat. Within amino acids 77-230, human RPTOR shares 100% sequence identity with mouse and rat RPTOR, making this region ideal for cross-species applications .

• Epitope location: Consider which domain of RPTOR you want to target. Different antibodies target distinct regions:

  • N-terminal regions (e.g., amino acids 90-140)

  • Central regions (e.g., amino acids 910-960)

  • C-terminal regions (e.g., amino acids 1140-1190)

• Clonality and host: Choose between monoclonal antibodies (higher specificity for a single epitope) and polyclonal antibodies (stronger signals by recognizing multiple epitopes). Common hosts include rabbit and mouse .

• Validation data: Review available data showing detection in specific cell lines (e.g., 293T, HeLa, Neuro-2A) and expected molecular weight (approximately 150 kDa, though some antibodies detect RPTOR at 68 kDa) .

What are optimal protocols for RPTOR antibody detection in Western blotting applications?

For optimal Western blot detection of RPTOR:

Sample Preparation:

• Use cell lysates from human or mouse cell lines known to express RPTOR (293T, HeLa, Neuro-2A, C2C12)

• Include protease inhibitors in lysis buffers to prevent degradation

• For phosphorylation studies, add phosphatase inhibitors

SDS-PAGE and Transfer:

• Use 6-8% gels due to RPTOR's high molecular weight (~150 kDa)

• Transfer to PVDF membrane for optimal protein binding

• Consider longer transfer times or lower current for efficient transfer of high molecular weight proteins

Antibody Incubation:

• Block with 5% non-fat dry milk or BSA in TBST

• Dilute primary RPTOR antibody according to manufacturer recommendations (typically 2-4 μg/mL)

• Incubate with appropriate HRP-conjugated secondary antibody (e.g., Anti-Mouse IgG Secondary Antibody)

Detection Considerations:

• Expect a band at approximately 150 kDa for full-length RPTOR, though some antibodies may detect it at different molecular weights (e.g., 68 kDa)

• Some antibodies perform optimally under non-reducing conditions

• Include positive control lysates and negative controls

Optimization Table:

How can flow cytometry protocols be optimized for RPTOR detection?

For successful flow cytometry detection of RPTOR:

Cell Preparation and Fixation:

• Since RPTOR is an intracellular protein, cells must be fixed and permeabilized

• Use Flow Cytometry Fixation Buffer followed by Flow Cytometry Permeabilization/Wash Buffer I

• Optimize fixation time (typically 10-20 minutes) to preserve epitope integrity while ensuring cell permeabilization

Antibody Staining:

• Use directly conjugated antibodies (e.g., Alexa Fluor® 488-conjugated or Alexa Fluor® 750-conjugated anti-RPTOR) for best results

• If using unconjugated primary antibodies, follow with appropriate fluorochrome-conjugated secondary antibodies

• Include isotype control antibody (e.g., MAB0041) to determine background staining levels

Controls and Validation:

• Include positive control cell lines with known RPTOR expression (HeLa, C2C12)

• Use negative controls: isotype controls and, when possible, RPTOR-knockdown cells

• For multicolor panels, include fluorescence minus one (FMO) controls

Analysis Considerations:

• Due to variable expression levels, adjust compensation settings appropriately

• Report median fluorescence intensity relative to isotype controls

• For phospho-RPTOR studies, compare both unstimulated and stimulated conditions

Experimental data has confirmed successful detection of RPTOR in HeLa human cervical epithelial carcinoma cells and C2C12 mouse myoblast cells using flow cytometry with both direct and indirect staining approaches .

What role does RPTOR play in disease models, and how can antibodies help elucidate these mechanisms?

RPTOR plays significant roles in multiple disease contexts:

Cancer Resistance Mechanisms:

RPTOR upregulation contributes to resistance against PI3K-mTOR inhibitors in renal cell carcinoma. Studies with the RCC4 cell line showed that cells resistant to BEZ235 (a dual PI3K-mTOR kinase inhibitor) overexpressed RPTOR at both mRNA and protein levels. This resistance was suppressed by RPTOR depletion or treatment with the allosteric mTORC1 inhibitor rapamycin . This suggests RPTOR expression should be monitored during PI3K-mTOR inhibitor therapy, with antibodies being essential tools for this assessment.

Neurodegenerative Diseases:

RPTOR has been identified as an Alzheimer's disease (AD) susceptibility gene. Recent genomic studies showed subjects homozygous for the major allele G of RPTOR SNP rs12940622 had increased prevalence of AD, while those with the minor allele A had decreased prevalence . The relationship between RPTOR and AD appears to involve complex interactions with BMI and mTOR signaling pathways. Antibody-based studies can help investigate RPTOR protein expression in neural tissues and correlate with disease progression.

Reproductive Disorders:

RPTOR is required for normal spermatogenesis. Research using germ cell-specific RPTOR knockout mice demonstrated that:

• RPTOR depletion leads to a significant reduction in germ cell numbers by postnatal day 8

• By P18, both undifferentiated (GFRA1+) and differentiating (KIT+) spermatogonia were dramatically reduced in knockout testes

• Adult RPTOR knockout mice developed a Sertoli cell-only phenotype with complete loss of germ cells

Data Table: RPTOR Genotype vs. Alzheimer's Disease (in subjects <55 years)

Subjects with the GG genotype showed significantly higher AD prevalence (p=0.05, Fisher's exact test) .

What strategies can be employed to design antibodies targeting specific epitopes within RPTOR?

Developing antibodies that target specific epitopes within RPTOR involves several advanced strategies:

Rational Design Approaches:

• Complementary Peptide Identification: Identify peptides that bind with high specificity and affinity to target regions of RPTOR. This can be achieved by analyzing amino acid sequence interactions in the Protein Data Bank (PDB) to identify potential interaction partners for a given target sequence .

• Antibody Scaffold Selection: Choose stable antibody scaffolds tolerant to peptide grafting. Human heavy chain variable (VH) domains that remain soluble and stable without light chain partners, and whose folding is insensitive to mutations in CDR loops, are excellent candidates .

• CDR Loop Engineering: Graft the complementary peptides onto CDR loops of the antibody scaffold. The third CDR (CDR3) loop is often the most suitable for peptide insertion .

• Multi-Loop Design Strategy: For higher affinity, engineer antibodies with complementary peptides grafted onto multiple CDR loops. These peptides can be designed to bind the target epitope cooperatively, creating a "pincer-like" binding mode .

Design Considerations:

• When selecting epitopes, disordered regions of RPTOR may be particularly amenable to this approach

• The complementary peptides must be carefully placed to avoid geometric constraints

• For two-loop designs, peptides should bind different sides of the same epitope without competing

Validation Methods:

• Verify binding specificity through multiple techniques (ELISA, Western blot, immunofluorescence)

• Test designed antibodies against both wild-type and RPTOR-depleted samples

• Evaluate cross-reactivity with related proteins to ensure specificity

This rational design approach has been successfully applied to create antibodies targeting disordered epitopes in disease-related proteins like α-synuclein, Aβ42, and IAPP, and similar principles could be applied to RPTOR .

How should I address unexpected molecular weight bands when using RPTOR antibodies?

When RPTOR antibodies detect bands at unexpected molecular weights (the calculated MW is 149 kDa, but bands may appear at different sizes):

Potential Causes:

• Multiple Isoforms: RPTOR has multiple isoforms that may be detected by the antibody. Some antibodies report detecting RPTOR at approximately 68 kDa despite the calculated MW of 149 kDa .

• Post-translational Modifications: Phosphorylation and other modifications can alter migration patterns. RPTOR contains multiple Ser/Thr residues subject to phosphorylation that regulates mTOR activation .

• Proteolytic Processing: RPTOR may undergo proteolytic cleavage during sample preparation or as part of cellular regulation.

• Experimental Conditions: Some antibodies require specific conditions (e.g., non-reducing) for optimal detection. The R&D Systems antibody (MAB5957) specifically detects RPTOR at 150 kDa under non-reducing conditions .

• Cross-reactivity: The antibody may detect proteins with similar epitopes. Review validation data showing specificity across different cell types.

Troubleshooting Approaches:

• Validate with Multiple Antibodies: Compare results using antibodies targeting different RPTOR epitopes.

• Include Appropriate Controls:

  • Positive controls: Lysates from cell lines known to express RPTOR (293T, HeLa, Neuro-2A)

  • Negative controls: RPTOR-depleted samples when available

• Optimize Sample Preparation:

  • Include protease inhibitors to prevent degradation

  • Test different lysis buffers to ensure complete extraction

  • Compare reducing vs. non-reducing conditions

• Perform Peptide Competition: Pre-incubate the antibody with its immunizing peptide to confirm specificity of observed bands.

• Consider Advanced Validation: For critical applications, confirm identity of detected bands by immunoprecipitation followed by mass spectrometry.

What is the relationship between RPTOR and the mTOR signaling pathway in antibody development?

The relationship between RPTOR and mTOR signaling extends into unexpected areas, including antibody development and immune responses:

Effect on Antibody Responses:

Research has shown that inhibiting mTOR with rapamycin (which affects the RPTOR-mTOR complex) during immunization with influenza virus promotes cross-strain protection against lethal infection with various influenza virus subtypes . This occurs through several mechanisms:

• Germinal Center Formation: Rapamycin reduces germinal center formation during the immune response .

• Class Switching Inhibition: The mTORC1 complex (containing RPTOR) is required for B cell class switching. Inhibiting this complex with rapamycin inhibits class switching, yielding a unique repertoire of antibodies .

• Antibody Specificity Shift: Rapamycin treatment skews the antibody response away from high-affinity variant epitopes and targets more conserved elements of viral proteins like hemagglutinin .

Implications for Research:

• RPTOR-Antibody Dual Research: When studying RPTOR using antibodies, researchers should consider how modulation of mTOR signaling (through drugs like rapamycin) might affect their experimental antibody-based detection systems.

• Antibody Development Applications: Understanding how RPTOR-mTOR signaling affects antibody responses could inform development of better antibodies for research and therapeutic purposes.

• Therapeutic Potential: Manipulation of RPTOR-mTOR signaling during vaccination could potentially create broader neutralizing antibody responses against pathogens with high antigenic drift.

• Experimental Design Considerations: When designing experiments involving both RPTOR detection and immune responses, consider how interventions affecting one might impact the other.

This connection between RPTOR-mTOR signaling and antibody responses highlights the importance of considering broader signaling contexts when using antibodies to study these pathways.

How can I validate the specificity of RPTOR antibodies for my experimental system?

Comprehensive validation of RPTOR antibodies requires multiple complementary approaches:

Genetic Validation:

• Knockdown/Knockout Controls: Use siRNA, shRNA, or CRISPR/Cas9 to reduce or eliminate RPTOR expression. The antibody signal should decrease proportionally .

  • Example: In studies of PI3K-mTOR inhibitor resistance, RAPTOR depletion confirmed antibody specificity while also demonstrating RAPTOR's role in resistance mechanisms .

• Overexpression Controls: Express tagged RPTOR constructs and confirm co-detection with your RPTOR antibody.

Biochemical Validation:

• Peptide Competition: Pre-incubate the antibody with its immunizing peptide. Signal reduction confirms epitope specificity .

  • Many commercial RPTOR antibodies offer blocking peptides for this purpose. For example, the immunizing peptide for certain antibodies is located within amino acids 90-140 or 1140-1190 of RPTOR .

• Multiple Antibodies: Test several antibodies targeting different RPTOR epitopes. Consistent results increase confidence in specificity.

• Molecular Weight Verification: Confirm detection at the expected molecular weight (~150 kDa for full-length RPTOR), while being aware that some antibodies detect RPTOR at different apparent molecular weights .

Application-Specific Validation:

• For Western Blotting:

  • Include positive control cell lysates (293T, HeLa, Neuro-2A, C2C12)

  • Compare reducing vs. non-reducing conditions

• For Immunofluorescence/IHC:

  • Compare staining patterns with multiple antibodies

  • Verify subcellular localization matches known RPTOR distribution

  • Include isotype control antibodies (e.g., MAB0041 for mouse monoclonals)

• For Flow Cytometry:

  • Include isotype controls to establish background levels

  • Compare staining in cells with manipulated RPTOR expression

Documentation and Reporting:

Maintain detailed records of validation experiments, including:

• Antibody catalog numbers and lot numbers

• Experimental conditions (fixation, blocking, dilutions)

• All controls used

• Images of full Western blots (not just the region of interest)

What advances in computational modeling are improving antibody design for targets like RPTOR?

Recent advances in computational modeling are transforming antibody design for targets like RPTOR:

Biophysics-Informed Modeling:

Computational approaches now integrate physical modeling with experimental data to improve antibody design. This involves:

• Binding Mode Identification: Advanced models can identify different binding modes associated with particular ligands, even when these ligands are chemically very similar .

• Energy Function Optimization: Computational methods optimize energy functions associated with each binding mode to generate antibodies with customized specificity profiles - either specific to a single target or cross-specific across multiple targets .

• Disentangling Complex Binding Modes: Modern algorithms successfully disentangle binding modes even when target epitopes cannot be experimentally isolated from other epitopes present in selection experiments .

Machine Learning Applications:

• Sequence-Based Prediction: Machine learning algorithms can predict antibody-antigen interactions based on sequence information alone, enabling the design of antibodies with desired binding properties .

• Library Design Optimization: Computational approaches can guide the design of smart antibody libraries with increased likelihood of yielding specific binders to targets like RPTOR.

• Experimental Data Integration: Models that combine high-throughput sequencing data with computational analysis enable the design of antibodies beyond those probed experimentally .

Practical Applications:

The combination of these approaches has enabled:

• Custom Specificity Profiles: Designing antibodies with either high specificity for a particular target or cross-specificity across multiple targets .

• Experimental Artifact Mitigation: Computational approaches help identify and mitigate experimental artifacts and biases in antibody selection experiments .

• Prediction of Optimal Experimental Conditions: Models can suggest optimal conditions for antibody performance, reducing the need for extensive empirical optimization.

These computational advances are particularly valuable for challenging targets like RPTOR that may have multiple conformations or exist in complex with other proteins.

How does RPTOR depletion affect the first wave of spermatogenesis, and how can antibodies help study this process?

RPTOR plays a crucial role in spermatogenesis, with its depletion causing significant defects:

Key Findings from Germ Cell-Specific RPTOR Knockout Studies:

• Reduced Germ Cell Population: Immunostaining for DDX4 (a pan germ cell marker) revealed a statistically significant twofold reduction in germ cells per testis cord in P8 Rptor knockout mice (P = 0.047) .

• Undifferentiated Spermatogonia Loss: By P8, there was a significant reduction in GFRA1+ and CDH1+ undifferentiated spermatogonia in knockout testes compared to wild-type .

• Progressive Depletion: By P18, both undifferentiated spermatogonia (GFRA1+) and differentiating spermatogonia (KIT+) were dramatically reduced (P = 0.007 and P = 0.0006, respectively) .

• Complete Germline Loss: By P60, knockout testes exhibited a Sertoli cell-only phenotype with complete absence of germ cells .

• Sertoli Cell Preservation: Numbers of GATA4+ Sertoli cells per testis cord were not statistically different between knockout and wild-type testes, indicating a germ cell-specific effect .

Antibody Applications in This Research:

Antibodies played a critical role in characterizing the RPTOR knockout phenotype:

• Cell Type Identification: Antibodies against specific markers allowed identification of cell populations:

  • DDX4/TRA98: Pan germ cell markers

  • GFRA1: Undifferentiated spermatogonia

  • CDH1: Undifferentiated spermatogonia

  • KIT: Differentiating spermatogonia

  • GATA4: Sertoli cells

  • MKI67: Proliferating cells

  • c-PARP1: Apoptotic cells

• Quantitative Analysis: Immunofluorescence with these antibodies enabled quantification of different cell populations:

  • The percentage of cells positive for each marker was calculated by dividing by the total number of germ cells labeled by DDX4 or TRA98

  • This allowed precise tracking of the progressive loss of specific spermatogonial populations

• Mechanistic Studies: Antibodies against phosphorylated proteins (e.g., p-AKT) helped investigate downstream signaling pathways affected by RPTOR deletion .

Research Implications:

This research demonstrated the germ cell-autonomous requirement for RPTOR during the first wave of spermatogenesis, particularly for completion of meiosis. The study highlights how antibodies enable detailed characterization of complex phenotypes in tissue-specific knockout models.

What considerations are important when designing experiments to study RPTOR phosphorylation states?

RPTOR phosphorylation is critical for regulating mTORC1 activity, requiring specific experimental design considerations:

Experimental Design Principles:

• Lysis Buffer Composition:

  • Include phosphatase inhibitors (sodium fluoride, sodium orthovanadate, β-glycerophosphate)

  • Use buffers that preserve phosphorylation (RIPA or specialized phospho-preservation buffers)

  • Process samples rapidly and maintain cold temperatures throughout

• Control Conditions:

  • Include phosphatase treatment controls to validate phospho-specific antibody detection

  • Compare phosphorylation under various conditions:

    • Nutrient starvation vs. nutrient-rich

    • Growth factor stimulation vs. withdrawal

    • Energy stress (e.g., AMPK activation)

    • mTOR inhibitors (rapamycin, mTOR kinase inhibitors)

• Antibody Selection:

  • Use phospho-specific antibodies targeting known RPTOR phosphorylation sites

  • Pair with total RPTOR antibodies whose epitopes are not affected by phosphorylation status

  • Validate antibody specificity with phosphatase-treated samples

• Detection Methods:

  • Western Blotting: Use dual-channel detection to simultaneously visualize phospho and total RPTOR

  • Immunoprecipitation: Pull down with total RPTOR antibody, then probe with phospho-specific antibodies

  • Immunofluorescence: Compare phospho-RPTOR localization under different conditions

  • Mass Spectrometry: For comprehensive phosphorylation site mapping

Key RPTOR Phosphorylation Sites and Functions:

Interpretive Framework:

• Calculate phospho-to-total RPTOR ratios rather than absolute phospho-signal

• Consider temporal dynamics of phosphorylation (immediate vs. sustained changes)

• Analyze phosphorylation in context of downstream mTORC1 targets (S6K, 4EBP1 phosphorylation)

• Be aware that different phosphorylation sites may have opposing effects on mTORC1 activity