NRAS Antibody

What types of NRAS antibodies are available for research purposes?

Several types of NRAS antibodies are available, each with specific characteristics suitable for different research applications:

Polyclonal antibodies (such as rabbit polyclonal ab167136) recognize multiple epitopes on the NRAS protein, providing strong signal amplification but potentially more background . These are typically generated against synthetic peptides corresponding to specific regions of human NRAS.

Monoclonal antibodies, including mouse monoclonals targeting N-terminal regions (70-101 amino acids), offer higher specificity for a single epitope, which can be advantageous for distinguishing between closely related proteins in the RAS family .

Mutation-specific antibodies like SP174 are specialized monoclonal antibodies designed to selectively recognize mutant forms of NRAS (specifically Q61R mutation), which is particularly valuable for cancer research and diagnostics .

What are the validated applications for NRAS antibodies in research?

While NRAS antibodies can theoretically be used across multiple immunoassay platforms, most commercially available options have been specifically validated for Western blotting (WB):

For Western blotting, NRAS antibodies detect the native protein at approximately 21 kDa . Some antibodies have been knockout validated, providing high confidence in their specificity .

For immunohistochemistry applications, mutation-specific antibodies like SP174 have been thoroughly validated for detecting NRAS Q61R mutations in formalin-fixed, paraffin-embedded tissue samples .

A comprehensive evaluation of 22 commercially available RAS antibodies revealed substantial variation in specificity and sensitivity, highlighting the importance of validation before experimental use .

How should NRAS antibodies be stored and handled to maintain optimal activity?

Proper storage and handling of NRAS antibodies is critical for maintaining their specificity and activity:

Short-term storage (1-2 weeks):

• Store at 4°C in a refrigerator

• Keep antibodies in their original buffer conditions

• Avoid repeated freeze-thaw cycles

Long-term storage:

• Store at -20°C in small aliquots

• For liquid formulations, maintain in the supplied buffer (typically PBS with 0.09% sodium azide as a preservative)

• Avoid exposure to light, especially for conjugated antibodies

Most NRAS antibodies are supplied in liquid form with preservation buffers:

• pH: 7.2 buffer conditions

• Preservative: 0.02% Sodium azide

• Constituent: 99% PBS

To minimize degradation, divide antibodies into small working aliquots before freezing to prevent repeated freeze-thaw cycles. Always centrifuge the vial briefly before opening to ensure all liquid is collected at the bottom of the vial.

What are the recommended protocols for using NRAS antibodies in Western blotting?

Optimizing Western blot protocols for NRAS detection requires attention to several technical parameters:

Key protocol considerations:

• Sample preparation:

  • Use appropriate lysis buffers containing protease inhibitors

  • Include positive control lysates (e.g., HEK-293, A549, MCF7 cells)

  • Consider knockout or knockdown controls where available

• Gel electrophoresis:

  • 10-12% SDS-PAGE gels are typically suitable for resolving the 21 kDa NRAS protein

  • Include molecular weight markers to confirm target band size

• Transfer and blocking:

  • PVDF or nitrocellulose membranes are both suitable

  • Block with 5% non-fat milk or BSA in TBS-T

• Antibody incubation:

  • Primary: Use manufacturer's recommended dilution (0.5-2 μg/ml or 1:1000)

  • Secondary: Match to primary antibody host species (anti-rabbit for rabbit polyclonal, anti-mouse IgG1 for mouse monoclonal)

• Detection:

  • ECL-based chemiluminescence is suitable for most NRAS antibody applications

  • Always verify that detected bands correspond to the expected 21 kDa molecular weight

How can researchers validate NRAS antibody specificity in their experimental systems?

Rigorous validation of NRAS antibodies is essential for reliable research results, particularly given the high sequence homology between RAS family members:

• Genetic validation:

  • Testing in cell lines with confirmed NRAS knockout or knockdown

  • Comparison with "Rasless" systems (cells lacking all RAS isoforms)

  • Testing in cells expressing only specific RAS isoforms

• Protein-level validation:

  • Western blot analysis against recombinant NRAS, KRAS, and HRAS proteins

  • Verification of expected molecular weight (21 kDa for NRAS)

  • Pre-absorption tests with specific blocking peptides

• Application-specific validation:

  • For Western blotting: Testing antibody performance across concentration gradients

  • For IHC/IF: Comparing staining patterns with known expression profiles

  • For mutation-specific antibodies: Correlation with genomic sequencing results

• Multi-antibody approach:

  • Using multiple antibodies targeting different epitopes of NRAS

  • Comparing results between different antibody clones

A systematic evaluation of RAS antibodies revealed that many antibodies do not perform as advertised, with some recognizing proteins other than their intended targets and others showing unexpected cross-reactivity patterns .

How do cross-reactivity issues with other RAS family proteins affect NRAS antibody selection?

Cross-reactivity between different RAS family members (HRAS, KRAS, NRAS) is a significant consideration when selecting NRAS antibodies:

The molecular basis for cross-reactivity stems from the high sequence homology (~85%) between RAS family proteins, with most differences concentrated in the C-terminal hypervariable region. Antibodies targeting conserved regions are more likely to cross-react.

To address cross-reactivity concerns:

• Select antibodies with demonstrated specificity through comprehensive validation

• Include appropriate controls (HRAS and KRAS expression, knockout models)

• Consider using multiple antibodies targeting different epitopes

• Validate findings with complementary techniques (RT-PCR, sequencing)

• For mutation-specific applications, confirm with genomic testing methods

How can mutation-specific NRAS antibodies be used to study oncogenic mutations?

Mutation-specific NRAS antibodies represent a powerful tool for studying oncogenic mutations at the protein level:

The SP174 antibody case study illustrates the principles and applications:

• Detection methodology:

  • SP174 selectively recognizes the NRAS Q61R mutation (resulting from c.182A>G substitution)

  • Immunohistochemistry protocol uses specific epitope retrieval (Leica H2 buffer, 25 minutes)

  • Optimal dilution (1:200) provides strong signal in positive controls with no background in negative controls

  • Semi-quantitative scoring system (0 to +3) allows standardized assessment

• Validation methodology:

  • Confirmation of mutation status by multiple methods:

    • Sanger sequencing

    • qPCR assay

    • Ion Torrent next-generation sequencing

• Cross-reactivity considerations:

  • SP174 shows cross-reactivity with KRAS Q61R mutations

  • This cross-reactivity must be considered when interpreting results

  • May be advantageous for pan-RAS Q61R mutation screening

Research applications include:

How do post-translational modifications affect NRAS antibody recognition?

Post-translational modifications (PTMs) of NRAS protein can significantly impact antibody recognition:

NRAS undergoes several important PTMs:

• Lipid modifications: NRAS is modified at its C-terminus by the addition of farnesyl or geranylgeranyl groups as part of the CAAX-signaled processing . These modifications enable membrane association, as indicated in the search results: "Cell membrane; Lipid-anchor; Cytoplasmic side. Golgi apparatus membrane; Lipid-anchor" .

• Processing state: The processing state of RAS proteins can influence antibody recognition: "asked whether Caax-signaled processing influenced recognition of KRAS proteins" . While this finding specifically refers to KRAS, similar principles likely apply to NRAS antibodies.

Key considerations for researchers:

• Epitope location: Antibodies targeting regions near modification sites may show differential binding depending on the modification state of NRAS:

  • N-terminal antibodies (targeting aa 70-101) may be less affected by C-terminal modifications

  • C-terminal antibodies might show differential recognition based on lipidation status

• Sample preparation impact: Extraction methods affect PTM preservation:

  • Detergent selection can disrupt lipid modifications

  • Phosphatase inhibitors preserve phosphorylation states

  • Buffer conditions affect protein conformation

• Subcellular localization: PTMs affect NRAS localization between plasma membrane, Golgi apparatus, and cytoplasm , influencing antibody accessibility in techniques like immunohistochemistry.

To ensure comprehensive detection regardless of PTM status, researchers should consider using multiple antibodies targeting different epitopes.

What are the methodological considerations for using NRAS antibodies in cancer research?

NRAS antibodies are particularly valuable in cancer research due to the frequent mutations of NRAS in various malignancies. Several methodological considerations are important:

• Mutation detection strategy:

  • Mutation-specific antibodies (e.g., SP174 for Q61R) provide direct protein-level detection

  • Always validate antibody results with genomic methods (Sanger sequencing, qPCR, NGS)

  • Consider the cross-reactivity pattern (SP174 detects both NRAS and KRAS Q61R mutations)

• Sample considerations:

  • Formalin-fixed paraffin-embedded (FFPE) tissues require optimized epitope retrieval conditions

  • Fresh-frozen samples may preserve epitopes better but have different processing requirements

  • Cell line studies should include appropriate positive and negative controls

• Technical parameters for IHC:

  • Antibody dilution optimization (1:200 for SP174)

  • Appropriate detection systems (e.g., polymer-based detection with diaminobenzidine)

  • Standardized scoring system (0 to +3)

• Interpretation challenges:

  • Intratumoral heterogeneity in mutation status

  • Distinguishing specific from non-specific staining

  • Correlation with clinical data and outcomes

• Multi-method approach:

  • Combine antibody-based detection with genomic analysis

  • Consider downstream signaling pathway activation

  • Integrate with functional studies when possible

For clinical research applications, standardization of protocols and interpretation criteria is essential to ensure reproducibility and comparability between studies.

What are common technical challenges when using NRAS antibodies and how can they be addressed?

Researchers commonly encounter several technical challenges when working with NRAS antibodies:

• Weak or absent signal in Western blotting:

  • Increase antibody concentration (within 0.5-2 μg/ml range for polyclonal, 1:1000 for monoclonal)

  • Extend primary antibody incubation time (overnight at 4°C)

  • Optimize protein loading (10-30 μg total protein)

  • Ensure target is expressed in sample (use positive controls: HEK-293, A549, MCF7)

  • Check transfer efficiency with reversible staining

• Non-specific bands:

  • Increase blocking stringency (5% BSA instead of milk)

  • Optimize antibody dilution

  • Include competing peptides to confirm specificity

  • Use gradient gels for better resolution around 21 kDa

  • Consider antibodies validated against knockout samples

• Background in immunohistochemistry:

  • Optimize blocking conditions

  • Titrate primary antibody (1:200 dilution for SP174)

  • Optimize antigen retrieval (Leica H2 buffer, 25 minutes for SP174)

  • Include appropriate negative controls

• Cross-reactivity with other RAS proteins:

  • Select highly validated isoform-specific antibodies (SC-31, SC-519)

  • Confirm findings with complementary techniques

  • Include RAS isoform controls when possible

  • Consider the specific epitope location relative to regions of sequence divergence

How can researchers distinguish between NRAS wild-type and mutant proteins?

Distinguishing between wild-type and mutant NRAS proteins requires specific methodological approaches:

• Mutation-specific antibodies:

  • SP174 antibody selectively detects NRAS Q61R mutant protein

  • Immunohistochemical approach using standardized protocols

  • Semi-quantitative scoring (0 to +3)

  • Important limitation: SP174 also recognizes KRAS Q61R mutations

• Combined genomic and protein approaches:

  • Validate antibody staining patterns with:

    • Sanger sequencing

    • qPCR assays targeting specific mutations

    • Next-generation sequencing (e.g., Ion Torrent)

  • Use cell lines with known mutation status as controls

• Functional assays:

  • Assess downstream pathway activation (phospho-ERK, phospho-AKT)

  • Combine with general NRAS antibodies to assess total vs. mutant expression

  • Consider GTP-binding assays (mutant RAS proteins often show impaired GTPase activity)

• Spatial analysis in tissue sections:

  • Compare mutation-specific antibody staining with total NRAS staining

  • Evaluate intratumoral heterogeneity

  • Correlate with histopathological features

• Technical considerations:

  • Optimize epitope retrieval conditions

  • Use appropriate detection systems

  • Include validated positive and negative controls

What considerations are important when comparing results across different NRAS antibodies?

When comparing results obtained using different NRAS antibodies, researchers should consider several factors:

• Target epitope differences:

  • N-terminal antibodies (aa 70-101) vs. C-terminal antibodies

  • Epitope accessibility in different applications

  • Influence of protein conformation on epitope exposure

• Antibody format differences:

  • Polyclonal antibodies recognize multiple epitopes, potentially providing stronger signal

  • Monoclonal antibodies offer higher specificity but may be more sensitive to epitope alterations

  • Isotype differences (IgG, IgG1) may affect secondary antibody selection

• Cross-reactivity patterns:

  • Differential recognition of HRAS, KRAS, and NRAS

  • Different antibodies show varying specificity profiles

  • Some antibodies claimed to be isoform-specific recognize unintended targets

• Application-specific performance:

  • Antibodies validated for Western blot may not perform well in IHC

  • Fixation and processing affect epitope accessibility differently

  • Buffer conditions may differentially impact antibody binding

• Standardization considerations:

  • Use consistent protocols when comparing antibodies

  • Include the same positive and negative controls

  • Consider quantitative approaches to assess relative performance

The comprehensive evaluation of 22 RAS antibodies demonstrated substantial variation in specificity and sensitivity, highlighting that manufacturer claims should be independently validated .