MRAS Antibody

What is the MRAS protein and why is it significant for research?

MRAS (muscle RAS oncogene homolog), also known as M-ras, RRAS3, or ras-related protein M-Ras, is a 23.8-24 kDa protein that functions as a signal transducer in the Ras-MAPK signaling pathway regulating cell proliferation and survival . It serves as a core component of the SHOC2-MRAS-PP1c (SMP) holophosphatase complex that regulates MAPK pathway activation . MRAS is particularly significant in research due to its role in the formation of the SMP complex when MRAS is GTP-bound, and its involvement in dephosphorylating inhibitory phosphorylation sites on RAF1, BRAF, and ARAF kinases, thereby stimulating their activities . Recent research has also linked MRAS mutations to Noonan syndrome with cardiac hypertrophy, establishing it as an important research target in developmental disorders .

What applications are MRAS antibodies typically used for in research?

MRAS antibodies are employed in multiple research applications including:

Researchers should note that each antibody may have optimal conditions that should be titrated in specific testing systems to obtain optimal results . The antibodies have demonstrated particular efficacy in detecting MRAS in brain tissue samples across species .

How do I select the appropriate MRAS antibody for my research?

When selecting an MRAS antibody, consider:

• Target specificity: Determine whether you need an antibody that recognizes specific domains or phosphorylation states of MRAS. Some antibodies target the C-terminal region while others may recognize full-length protein .

• Host species and antibody type: MRAS antibodies are available as rabbit polyclonal, rabbit monoclonal, and other formats. Consider potential cross-reactivity issues based on your experimental model .

• Validated applications: Verify that the antibody has been validated for your intended application (WB, IP, IF, IHC) .

• Species reactivity: Ensure the antibody recognizes MRAS in your species of interest. Available antibodies show reactivity with human, mouse, and rat samples .

• Published validation data: Review validation galleries and publications that have successfully used the antibody for similar applications .

The extensive range of available products (over 200 across multiple suppliers) necessitates careful review of validation data before selection .

What are the optimal sample preparation methods for MRAS detection by Western blot?

For optimal MRAS detection by Western blot:

• Tissue selection: Brain and lung tissues consistently show detectable levels of MRAS expression .

• Lysis buffer composition: For MRAS protein extraction, use buffer with adequate detergent concentration (typically containing NP-40 or Triton X-100) to solubilize membrane-associated proteins.

• Protein amount: Load 20-40 μg of total protein lysate per lane for cell lines and 30-50 μg for tissue samples.

• Antibody dilution: For established antibodies like 14213-1-AP, use at 1:500-1:1000 dilution . For other antibodies, follow manufacturer recommendations or optimize through titration.

• Blocking conditions: 5% non-fat milk or 3-5% BSA in TBST is typically effective.

• Detection system: Enhanced chemiluminescence (ECL) systems provide sufficient sensitivity for MRAS detection in most samples.

• Controls: Include positive controls (known MRAS-expressing tissues like brain) and negative controls (tissues with low MRAS expression or MRAS-knockout samples).

The expected molecular weight for MRAS is 24 kDa, which should be confirmed during analysis .

How can I optimize MRAS immunoprecipitation protocols for protein-protein interaction studies?

For optimized MRAS immunoprecipitation in protein-protein interaction studies:

• Antibody selection: Choose antibodies validated for IP applications, such as those that have demonstrated successful IP in mouse brain tissue .

• Antibody amount: Use 0.5-4.0 μg of antibody per 1.0-3.0 mg of total protein lysate .

• Lysis conditions: Use mild lysis buffers containing 0.5-1% NP-40 or Triton X-100 to preserve protein-protein interactions.

• Pre-clearing: Pre-clear lysates with protein A/G beads to reduce non-specific binding.

• Incubation conditions: Incubate antibody with lysate overnight at 4°C with gentle rotation.

• Washing stringency: Employ multiple washes with decreasing detergent concentrations to remove non-specific interactions while preserving genuine interactions.

• Elution conditions: For functional assays, consider native elution using competitive peptides; for mass spectrometry analysis, use more denaturing conditions.

• Controls: Always include IgG control immunoprecipitations and input samples for comparison.

When studying MRAS in the context of the SMP complex, consider cross-linking approaches to stabilize transient interactions .

What are the key considerations for studying MRAS expression in disease models using antibody-based methods?

When studying MRAS expression in disease models:

• Model selection: For Noonan syndrome research, cardiac and developmental models are particularly relevant given MRAS's role in cardiac hypertrophy .

• Expression level changes: Monitor both total MRAS expression and activation state (GTP-bound form).

• Spatial distribution: Use immunohistochemistry to track tissue-specific expression patterns, particularly in brain, heart, and developmental tissues .

• Temporal dynamics: Plan time-course experiments to capture dynamic changes in MRAS expression during disease progression.

• Parallel signaling pathways: Concurrently assess related proteins in the Ras-MAPK pathway using multiplexed approaches:

• Experimental controls: Include appropriate genotypic controls (wild-type vs. mutant) and treatment controls (pathway inhibitors or activators) .

• Validation approaches: Validate antibody-based findings with complementary techniques like RNA expression analysis or genetic manipulation .

How do I address non-specific binding or background issues when using MRAS antibodies?

When encountering non-specific binding or high background:

• Antibody validation: Verify antibody specificity using positive and negative controls, including MRAS-knockout or knockdown samples.

• Blocking optimization: Test different blocking agents (BSA, non-fat milk, commercial blockers) and concentrations (3-5%).

• Antibody dilution: Further dilute the primary antibody; for example, extend the range from 1:500-1:1000 to 1:2000-1:5000 if signal is strong .

• Washing protocol enhancement: Increase washing duration or number of washes using TBS-T (0.1-0.3% Tween-20).

• Secondary antibody considerations: Use highly cross-adsorbed secondary antibodies and consider fluorescent secondaries for more quantitative results.

• Sample preparation improvements: For tissues with high lipid content (e.g., brain), additional delipidation steps may reduce background.

• Epitope competition: If available, pre-incubate the antibody with the immunizing peptide as a specificity control.

Non-specific bands may appear in Western blots of brain tissues due to splice variants or post-translational modifications. These patterns should be documented and consistent between experiments .

How can I distinguish between specific MRAS signals and cross-reactivity with other RAS family proteins?

To distinguish MRAS signals from other RAS family proteins:

• Antibody epitope selection: Prefer antibodies generated against unique regions of MRAS that have minimal homology with other RAS family members .

• Expression pattern analysis: Compare the expression pattern with known tissue distribution of MRAS (high in brain and lung) versus other RAS proteins .

• Molecular weight differentiation: MRAS has a distinctive molecular weight of 24 kDa, which can help differentiate it from other RAS proteins .

• Genetic validation: Use siRNA/shRNA knockdown or CRISPR knockout of MRAS to confirm specificity.

• Immunodepletion experiments: Sequential immunoprecipitation with antibodies against different RAS proteins can help identify cross-reactivity.

• Parallel analysis: Run samples on parallel blots probed with antibodies specific to different RAS family members.

• Activated state detection: Use GTP-loading assays specific for MRAS to distinguish functional activity from other RAS proteins.

The sequence homology between MRAS and other RAS family members requires rigorous validation to ensure specificity of signals, particularly in systems where multiple RAS proteins are expressed .

What strategies should I use to quantify MRAS expression or activation levels accurately?

For accurate quantification of MRAS expression or activation:

• Standard curve generation: Include recombinant MRAS protein standards for absolute quantification.

• Internal loading controls: Use housekeeping proteins (β-actin, GAPDH) appropriate for your tissue/cell type.

• Normalization approaches:

  • For total MRAS: Normalize to loading controls and total protein

  • For activated MRAS: Express as ratio of GTP-bound to total MRAS

• Quantitative techniques: Consider these approaches:

• Activation-specific measurements: For GTP-bound MRAS, use active RAS pull-down assays with GST-RBD (RAS-binding domain).

• Technical replicates: Include at least three technical replicates for statistical robustness.

• Biological replicates: Analyze multiple independent biological samples (different animals/cell preparations).

• Dynamic range verification: Ensure measurements fall within the linear range of detection.

Targeted mass spectrometry approaches have been developed for RAS network proteins and may provide more accurate quantification for challenging samples .

How can I effectively use MRAS antibodies to study the composition and dynamics of the SHOC2-MRAS-PP1c (SMP) holophosphatase complex?

To study the SMP complex effectively:

• Co-immunoprecipitation optimization: Use antibodies against MRAS to pull down the complex, then probe for SHOC2 and PP1c:

  • Lysis buffer: Use buffers that preserve complex integrity (low detergent, physiological salt)

  • Cross-linking: Consider reversible cross-linking to stabilize transient interactions

  • GTP-loading state: Compare complex formation under different nucleotide-loading conditions

• Proximity ligation assays (PLA): Detect in situ protein-protein interactions between MRAS and SHOC2 or PP1c in intact cells.

• FRET/BRET approaches: Monitor real-time complex dynamics using fluorescence/bioluminescence resonance energy transfer between tagged components.

• Immunofluorescence co-localization: Track subcellular distribution of complex components under different cellular conditions.

• Antibody accessibility experiments: Probe whether antibody epitopes become masked when the complex forms.

• Mutational analysis: Compare complex formation between wild-type and mutant MRAS (e.g., G23V mutation associated with Noonan syndrome) .

• Phosphatase activity correlation: Link complex formation to dephosphorylation of RAF kinases at specific inhibitory sites (Ser-259 of RAF1, Ser-365 of BRAF, Ser-214 of ARAF) .

Understanding this complex is particularly important since MRAS mutations that affect SMP complex formation have been linked to human developmental disorders .

What approaches can be used to investigate the role of MRAS in Noonan syndrome using antibody-based methods?

For investigating MRAS in Noonan syndrome:

• Patient-derived samples: Analyze MRAS expression and activation in:

  • Lymphoblastoid cell lines from patients

  • iPSC-derived cardiomyocytes harboring MRAS mutations

  • Animal models expressing MRAS mutations

• Mutation-specific assays: Compare wild-type versus mutant (e.g., G23V) MRAS:

  • Protein stability assessment

  • GTP-binding capacity

  • Interaction propensity with partner proteins

  • Subcellular localization patterns

• Downstream signaling analysis: Monitor phosphorylation of RAF kinases and ERK1/2 using phospho-specific antibodies:

  • Basal activity levels

  • Response to growth factor stimulation

  • Recovery kinetics after pathway inhibition

• Cardiac phenotype correlation: Link molecular findings to cardiac abnormalities:

  • Cardiomyocyte hypertrophy markers

  • Sarcomere organization

  • Ca²⁺ handling proteins

• Combinatorial antibody applications: Use multiple antibody-based methods:

• Developmental staging: Track MRAS expression and activity through developmental stages relevant to Noonan syndrome manifestations .

• Therapeutic monitoring: Use antibody-based assays to evaluate responses to targeted therapies (e.g., MEK inhibitors).

These approaches can help establish genotype-phenotype correlations and identify potential therapeutic targets for MRAS-mediated Noonan syndrome .

How can mass spectrometry complement antibody-based detection of MRAS in complex biological samples?

Mass spectrometry and antibody approaches can be integrated for comprehensive MRAS analysis:

• Immunoprecipitation-mass spectrometry (IP-MS):

  • Use MRAS antibodies to enrich for MRAS and interacting partners

  • Identify novel interaction partners or post-translational modifications

  • Quantify low-abundance MRAS in complex samples

• Targeted mass spectrometry approaches:

  • Selected/Multiple Reaction Monitoring (SRM/MRM) for MRAS peptides

  • Parallel Reaction Monitoring (PRM) for increased specificity

  • SWATH-MS for comprehensive pathway analysis

• Immuno-MRM assays:

  • Combine antibody enrichment with MRM-MS for enhanced sensitivity

  • Enable detection of multiple MRAS peptides and phosphopeptides simultaneously

  • Provide absolute quantification when using isotope-labeled standards

• PTM mapping:

  • Identify novel MRAS modifications beyond what antibodies can detect

  • Monitor multiple modification sites simultaneously

  • Discover potential regulatory mechanisms

• Verification of antibody specificity:

  • Confirm that antibody-detected bands contain MRAS peptides

  • Identify potential cross-reacting proteins

• Integrative workflows:

The RAS Initiative has developed a suite of antibody reagents that work synergistically with mass spectrometry approaches for comprehensive analysis of RAS network proteins, including MRAS .

What are the current challenges and emerging solutions in developing highly specific monoclonal antibodies against MRAS?

Current challenges and emerging solutions include:

• Epitope selection challenges:

  • High homology between RAS family members limits unique epitopes

  • Solutions: Focus on hypervariable regions or conformational epitopes specific to MRAS

• Isoform and modification specificity:

  • Detecting specific modifications (e.g., GTP-bound state)

  • Solutions: Generate conformation-specific antibodies using stabilized MRAS-GTP as immunogen

• Production challenges:

  • Traditional hybridoma methods have limited epitope coverage

  • Solutions:

    • Rapid generation techniques using vaccination-derived ASCs (antibody-secreting cells)

    • Phage display with synthetic antibody libraries

• Validation requirements:

  • Need for extensive cross-reactivity testing

  • Solutions:

    • Systematic validation across multiple applications following community-developed guidelines

    • CRISPR knockout validation systems

• Emerging technologies:

• Novel formats:

  • Bispecific antibodies targeting MRAS and binding partners

  • Intrabodies that can detect active MRAS inside living cells

  • Antibody-based biosensors for real-time MRAS activation monitoring

• Reproducibility considerations:

  • Batch-to-batch variation affects reliability

  • Solutions: Recombinant antibody production with sequence verification

The RAS Initiative's systematic approach to antibody development provides a model for generating well-validated reagents for studying MRAS and other RAS family proteins .

How might antibody-based approaches contribute to therapeutic targeting of MRAS in RAS-driven diseases?

Antibody-based approaches for therapeutic targeting of MRAS include:

• Diagnostic companion tools:

  • Antibodies that detect MRAS activation state

  • Patient stratification based on MRAS expression patterns

  • Monitoring treatment response in clinical trials

• Intracellular antibody fragments:

  • Cell-penetrating antibody fragments that disrupt MRAS-effector interactions

  • Transbodies targeting specific conformations of MRAS

  • Intrabodies expressed from gene therapy vectors

• Targeted protein degradation:

  • PROTAC-antibody conjugates to induce MRAS degradation

  • Lysosome-targeting antibody conjugates

• Disruption of the SMP complex:

  • Antibodies or derived fragments that interfere with MRAS-SHOC2 or MRAS-PP1c interactions

  • Targeting the GTP-bound state that enables complex formation

• Combination therapy approaches:

  • Antibody tools to identify synergistic targets in the MAPK pathway

  • Monitoring compensatory mechanisms during RAS-targeted therapy

• Technology integration:

• Developmental disorder applications:

  • Correcting aberrant signaling in Noonan syndrome models

  • Preventing cardiac hypertrophy in MRAS mutation carriers

While direct targeting of intracellular MRAS remains challenging, antibody tools continue to advance our understanding of therapeutic vulnerabilities in MRAS-driven diseases .

What novel techniques are emerging for studying MRAS localization and trafficking using antibody-based imaging?

Emerging techniques for MRAS imaging include:

• Super-resolution microscopy:

  • STORM/PALM imaging with MRAS antibodies for nanoscale localization

  • SIM for dynamic membrane interactions of MRAS

  • Lattice light-sheet microscopy for 3D visualization of MRAS trafficking

• Live-cell imaging approaches:

  • Fluorescent intrabodies (genetically encoded antibody fragments)

  • Antibody-based biosensors that change fluorescence upon MRAS activation

  • SNAP/CLIP-tag fusions validated with antibody colocalization

• Spatial omics integration:

  • Combining in situ hybridization with MRAS immunofluorescence

  • Correlative light and electron microscopy for ultrastructural context

  • Multiplexed ion beam imaging (MIBI) for simultaneous detection of dozens of MRAS pathway components

• Advanced tissue analysis:

  • Tissue clearing techniques with MRAS antibody penetration

  • Expansion microscopy for physical magnification of MRAS-containing structures

  • 3D organ-wide mapping of MRAS distribution

• Proximity-based methods:

  • BioID or APEX2 proximity labeling validated with antibody detection

  • Split-fluorescent protein complementation verified with MRAS antibodies

  • Three-dimensional interaction mapping in intact cells

• Quantitative approaches:

• Clinical imaging translation:

  • Radioimmunoconjugates for visualizing MRAS-driven tumors

  • Intraoperative fluorescence guidance based on MRAS pathway activation

These emerging technologies will enable researchers to understand the dynamic behavior of MRAS in normal physiology and disease states with unprecedented resolution .

What are the key quality control parameters that should be assessed when validating a new MRAS antibody for research use?

Comprehensive MRAS antibody validation should include:

• Specificity assessments:

  • Western blotting against recombinant MRAS and related RAS proteins

  • Testing in MRAS knockout/knockdown models

  • Peptide competition assays

  • Immunoprecipitation-mass spectrometry verification

• Sensitivity measurements:

  • Detection limit determination with purified protein

  • Signal-to-noise ratio in relevant biological samples

  • Comparison with established MRAS antibodies

• Application-specific validation:

• Cross-reactivity testing:

  • Against other RAS family members (HRAS, KRAS, NRAS, RRAS)

  • In tissues known to express multiple RAS proteins

  • Across species (if claiming multi-species reactivity)

• Reproducibility assessment:

  • Lot-to-lot consistency

  • Inter-laboratory testing

  • Performance stability over time/storage conditions

• Conformational specificity:

  • Testing against active (GTP-bound) vs. inactive (GDP-bound) MRAS

  • Detection of native vs. denatured protein

• Documentation standards:

  • Complete information on immunogen

  • Validation methods described in detail

  • Raw data availability

  • Application-specific recommendations

The RAS Initiative has established comprehensive validation protocols that can serve as a model for MRAS antibody validation across multiple applications .

How should researchers integrate antibody-based and genetic approaches when studying MRAS function in different experimental systems?

Effective integration of antibody and genetic approaches:

• Complementary validation strategy:

  • Use genetic tools (CRISPR, RNAi) to validate antibody specificity

  • Use antibodies to confirm genetic manipulation effects on protein levels

  • Combine approaches to distinguish transcript vs. protein level regulation

• Functional dissection workflow:

• Temporal control considerations:

  • Inducible genetic systems for defined timing

  • Antibody detection for monitoring kinetics

  • Combined approaches for validating immediate vs. delayed effects

• Spatial resolution integration:

  • Tissue-specific genetic manipulation

  • Immunohistochemical detection of resulting changes

  • Correlative approaches for phenotype-expression relationships

• Translational applications:

  • Patient-derived models with genetic characterization

  • Antibody-based profiling of pathway activation

  • Integrated biomarker development

• Pathway analysis:

  • Genetic manipulation of MRAS

  • Antibody detection of downstream signaling changes

  • Multi-level validation of pathway connections

• Therapeutic target validation:

  • Genetic proof-of-concept

  • Antibody-based mechanism confirmation

  • Combined approaches for identifying resistance mechanisms

This integrated approach has been particularly valuable in establishing MRAS's role in developmental disorders like Noonan syndrome with cardiac hypertrophy .

What methodological considerations should researchers address when comparing data generated using different MRAS antibodies across studies?

When comparing data from different MRAS antibodies:

• Epitope mapping considerations:

  • Document which domain/region each antibody targets

  • Consider how different epitopes affect detection in various states

  • Evaluate whether epitopes are conserved across species being compared

• Methodological standardization:

  • Note differences in sample preparation protocols

  • Account for variations in detection methods

  • Consider differences in quantification approaches

• Antibody format influences:

• Validation consistency assessment:

  • Compare validation methods used for each antibody

  • Identify potential blind spots in validation

  • Consider supplementary validation when combining datasets

• Batch effect handling:

  • Implement statistical methods to account for batch effects

  • Include common reference samples across experiments

  • Consider meta-analysis approaches for heterogeneous datasets

• Application-specific considerations:

  • WB: Different lysis methods may extract MRAS differently

  • IHC: Fixation and antigen retrieval variations affect detection

  • IP: Buffer conditions influence interactions detected

• Reporting standards implementation:

  • Use standardized antibody identifiers (RRID)

  • Document complete methodological details

  • Share raw data when possible