NF1 Antibody, HRP conjugated

What is NF1 and why are NF1 antibodies important in research?

Neurofibromin 1 (NF1) is a tumor suppressor protein encoded by the NF1 gene, mutations in which cause Neurofibromatosis type 1, an autosomal dominant genetic disorder. Nearly 3000 different disease-causing variants within the NF1 gene have been identified in the Human Gene Mutation Database, with up to 44% causing splicing errors in pre-mRNA . NF1 functions as a GTPase-activating protein (GAP) that negatively regulates Ras activity and also regulates adenylyl cyclase (AC) activity and cAMP levels .

NF1 antibodies enable researchers to:

• Detect NF1 protein expression in various tissues and cell types

• Investigate effects of NF1 mutations on protein function

• Study NF1's role in Ras and adenylyl cyclase signaling pathways

• Validate therapeutic approaches, such as antisense oligomers for correcting splicing defects

• Track changes in NF1 expression and localization under different experimental conditions

What applications are NF1 antibodies commonly used for?

NF1 antibodies demonstrate versatility across multiple research applications:

• Western blot (WB): For detecting NF1 protein expression levels in cell or tissue lysates, typically revealing a specific band at approximately 319 kDa

• Immunohistochemistry (IHC): For visualizing NF1 distribution in tissue sections, including cancer tissues such as mammary, rectal, bladder, and colorectal carcinomas

• Immunocytochemistry (ICC): For examining NF1 subcellular localization in cultured cells

• Immunofluorescence (IF): For high-resolution imaging of NF1 distribution

• Flow cytometry: For quantifying NF1 expression across cell populations

• ELISA: For quantitative measurement of NF1 in solution

These applications allow researchers to comprehensively characterize NF1 expression, function, and interaction with other cellular components.

What are the optimal experimental conditions for using NF1 antibodies?

Based on validated protocols for NF1 antibody applications, the following conditions yield optimal results:

For Western blot:

• Use 5-20% SDS-PAGE gel to properly resolve the large NF1 protein (~319 kDa)

• Load 50 μg of protein sample under reducing conditions

• Transfer to nitrocellulose membrane at 150 mA for 50-90 minutes

• Block with 5% non-fat milk in TBS for 1.5 hours at room temperature

• Incubate with anti-NF1 antibody at 0.5 μg/mL overnight at 4°C

• Wash thoroughly with TBS-0.1% Tween (3 times, 5 minutes each)

• Use enhanced chemiluminescent detection systems for optimal sensitivity

For IHC applications:

• Perform heat-mediated antigen retrieval in EDTA buffer (pH 8.0)

• Block tissue sections with 10% goat serum

• Apply primary NF1 antibody at 2 μg/ml concentration overnight at 4°C

• Use HRP-conjugated secondary antibody with DAB as chromogen for visualization

For flow cytometry:

• Fix cells with 4% paraformaldehyde

• Permeabilize membranes for intracellular staining

• Block with 10% normal goat serum

• Use 1 μg antibody per 1×10^6 cells

What controls should be included when using NF1 antibodies?

Including appropriate controls is essential for generating reliable data with NF1 antibodies:

• Positive controls: Samples known to express NF1 (e.g., HeLa cells as demonstrated in Western blot validation)

• Negative controls: For flow cytometry, unlabelled samples without primary and secondary antibody incubation establish baseline signals

• Isotype controls: Matched isotype antibody (e.g., rabbit IgG for rabbit anti-NF1) to assess non-specific binding, particularly important in flow cytometry applications

• Loading controls: Housekeeping proteins to normalize protein loading in Western blots

• Expression validation controls: When studying NF1 mutations, wild-type samples provide crucial comparison points for assessing changes in expression or function

• Functional controls: When studying NF1's role in signaling pathways, treatments that modulate these pathways (e.g., growth factors) help validate antibody specificity in detecting functional changes

How should I optimize sample preparation for NF1 detection?

Successful NF1 detection requires careful sample preparation tailored to the specific application:

For protein extraction and Western blot:

• Lyse cells with RIPA buffer

• Clear lysates by centrifugation at 20,000 RPM for 20 min at 4°C

• Quantify protein using Bradford assay

• For NF1 blots, load 50 μg of protein per well (higher than the 10 μg typically used for other proteins)

• Run 8% SDS-polyacrylamide gels at 100 V for 2 hours

• Transfer to PVDF membrane at 100 V for 2 hours

For tissue sections:

• Perform heat-mediated antigen retrieval in EDTA buffer (pH 8.0)

• Block tissue sections with 10% goat serum before antibody incubation

For flow cytometry:

• Fix cells with 4% paraformaldehyde

• Use permeabilization buffer to facilitate intracellular staining

• Block with 10% normal goat serum

These optimized preparation methods ensure maximum NF1 antigen accessibility while preserving sample integrity.

How do I address the challenges of detecting large proteins like NF1?

NF1's large size (~319 kDa) presents specific challenges for detection:

• Gel selection: Use gradient gels (5-20% SDS-PAGE) or lower percentage gels (8%) to adequately resolve high molecular weight proteins

• Extended transfer times: Increase transfer time (2 hours at 100V) to ensure complete protein transfer to membranes

• Increased protein loading: Use higher protein amounts (50 μg) compared to standard Western blot protocols

• Optimized antibody concentration: Use validated concentrations (0.5 μg/mL for Western blot, 2 μg/ml for IHC) to achieve optimal signal-to-noise ratio

• Sensitive detection systems: Employ enhanced chemiluminescent detection systems to visualize potentially weak signals from large proteins

• Protease inhibitors: Include comprehensive protease inhibitor cocktails during sample preparation to prevent degradation of the large NF1 protein

• Optimal incubation conditions: Increase primary antibody incubation time (overnight at 4°C) to ensure sufficient binding

How can I validate the specificity of NF1 antibodies in my experimental system?

Rigorous validation ensures reliable results when using NF1 antibodies:

• Molecular weight verification: Confirm detection of a band at the expected molecular weight (~319 kDa) in Western blot applications

• Multiple applications: Validate antibody performance across different techniques (Western blot, IHC, flow cytometry) as demonstrated in search result

• Multiple tissue types: Test antibody in various tissue contexts to ensure consistent staining patterns, as shown across mammary, rectal, bladder, and colorectal cancer tissues

• Correlation with functional assays: Verify that antibody-detected changes in NF1 expression correlate with functional outcomes, such as changes in Ras signaling or adenylyl cyclase activity

• Genetic models: Use NF1 mutant models to validate antibody sensitivity in detecting alterations in protein expression or function

• RNA-protein correlation: Compare protein detection results with mRNA expression data to confirm consistency

• Signal manipulation: Use treatments known to affect NF1 levels (e.g., PMO treatment as in search result ) to confirm the antibody can detect expected changes

What factors affect the performance of HRP-conjugated NF1 antibodies?

Several factors influence the performance of HRP-conjugated NF1 antibodies:

• Storage conditions: Maintain at -20°C for lyophilized antibody; after reconstitution, store at 4°C for one month or aliquot and freeze at -20°C for up to six months; avoid repeated freeze-thaw cycles

• Substrate selection: Different substrates (DAB, TMB, luminol-based) affect sensitivity and signal-to-noise ratio

• Incubation time: Optimize substrate incubation time to maximize specific signal while minimizing background

• Buffer composition: Ensure buffers are freshly prepared and at optimal pH for HRP activity

• Blocking effectiveness: Insufficient blocking leads to high background; optimize blocking conditions (5% non-fat milk, 10% serum) based on application

• Sample preparation: Inadequate fixation or permeabilization can reduce antibody accessibility to target epitopes

• Environmental factors: Temperature, light exposure, and oxidizing agents can affect HRP enzyme activity and stability

Optimizing these factors helps maximize sensitivity and specificity when using HRP-conjugated NF1 antibodies.

How can NF1 antibodies be used to study NF1 mutations and their effects?

NF1 antibodies enable detailed investigation of mutation effects on protein function:

How can NF1 antibodies contribute to understanding NF1's role in Ras signaling pathways?

NF1 antibodies provide crucial tools for investigating NF1's regulatory role in Ras signaling:

• RasGAP activity assessment: Monitor how NF1 mutations affect its ability to downregulate Ras. Mutations R1276P, R1391S, and K1423E were shown to reduce or abolish GAP activity

• Downstream signaling effects: Track changes in MAPK pathway activation. Wild-type hNF1 reduced phospho-MAPK to normal levels in Nf1 mutant flies, while mutants with defective RasGAP activity (R1276P, R1391S, K1423E) or lacking the GRD could not reduce phospho-MAPK levels

• Domain functionality studies: Determine which domains are sufficient for Ras regulation. GRD fragments alone (GRD1, GRD2) were able to restore phospho-MAPK to wild-type levels, indicating the sufficiency of this domain for Ras regulation

• Mutation comparison studies: Compare different mutations' effects on Ras pathway regulation. The L847P mutation did not affect the RasGAP activity of full-length hNF1, unlike other mutations

• Therapeutic validation: Assess correction of aberrant Ras signaling by treatments. PMO treatment targeting the cryptic splice site in the Y489C variant restored neurofibromin's ability to repress pERK/ERK and GTP-Ras levels

What approaches can reveal NF1's function in adenylyl cyclase activation?

NF1 plays a critical role in adenylyl cyclase (AC) regulation, which can be investigated using these approaches:

• Growth factor response studies: Examine how NF1 mutations affect growth factor-stimulated AC activity. RasGAP activity was shown to be required for EGF-stimulated AC activity, as mutant hNF1s with defective RasGAP activity or lacking the GRD did not respond to EGF stimulation

• Neurotransmitter response analysis: Determine NF1's role in mediating neurotransmitter effects on AC. Serotonin- and histamine-stimulated AC activity was examined to investigate whether RasGAP activity is required for the NF1/Gα-dependent AC pathway

• Domain mapping: Identify which NF1 domains are critical for AC regulation. The GRD fragments responded normally to EGF, indicating that the RasGAP activity of the GRD is required for growth factor-stimulated NF1/Ras-dependent AC activity

• Mutation effect characterization: Compare how different mutations impact AC activation. Various NF1 missense mutations and partial deletions were examined for their effects on growth factor and neurotransmitter-stimulated AC activity

• Cross-species conservation studies: Explore evolutionary conservation of NF1's AC regulatory function. Human NF1 was shown to function in Drosophila Nf1 mutants, demonstrating conservation of this regulatory pathway

How can NF1 antibodies help investigate therapeutic approaches for NF1-related disorders?

NF1 antibodies play a critical role in developing and validating therapeutic strategies:

• Antisense oligomer evaluation: Assess correction of splicing defects by antisense approaches. PMO treatment targeting the cryptic splice site created by the c.1466A>G variant restored normal splicing, as confirmed using NF1 antibodies to detect protein restoration

• Functional restoration measurement: Determine whether treatments restore normal protein function. PMO M1 treatment restored approximately 30% of wild-type NF1 protein levels, which was sufficient to restore signaling functions

• Dosage optimization: Determine minimum effective treatment dosages by quantifying protein restoration at different treatment concentrations

• Long-term efficacy assessment: Monitor persistence of therapeutic effects over time by tracking NF1 protein levels after treatment

• Mechanism validation: Confirm therapeutic mechanisms. For the Y489C variant, antibody detection showed that correcting the aberrant splicing successfully restored protein expression, confirming that the missense variant alone does not disrupt function

• Comparative approach evaluation: Compare different therapeutic strategies by measuring their relative efficiency in restoring NF1 protein levels and function

What causes inconsistent or weak signals when using NF1 antibodies?

Several factors can contribute to suboptimal NF1 detection:

• Protein degradation: NF1's large size makes it susceptible to degradation. Use fresh samples with comprehensive protease inhibitor cocktails during preparation

• Insufficient protein loading: NF1 detection requires higher protein amounts (50 μg) compared to many other proteins (10 μg)

• Inadequate transfer: Large proteins transfer less efficiently. Optimize transfer conditions (100V for 2 hours) for complete transfer to membranes

• Suboptimal antibody concentration: Titrate antibody concentration; recommended ranges are 0.5 μg/mL for Western blot and 2 μg/ml for IHC

• Ineffective antigen retrieval: For IHC/ICC, optimize heat-mediated antigen retrieval using EDTA buffer (pH 8.0)

• Improper storage: Antibody activity can diminish with improper storage or repeated freeze-thaw cycles

• Insufficient incubation time: Extend primary antibody incubation to overnight at 4°C to improve signal strength

• Detection system sensitivity: Use enhanced chemiluminescent detection systems for optimal visualization of potentially weak signals

How can I distinguish between specific and non-specific signals?

Differentiating genuine NF1 signals from artifacts requires multiple validation approaches:

• Molecular weight verification: Confirm detection at the expected 319 kDa size for full-length NF1

• Control samples: Include positive controls (e.g., HeLa cells), negative controls, and isotype controls (rabbit IgG for rabbit anti-NF1)

• Signal pattern analysis: Evaluate whether the cellular or tissue distribution pattern matches known NF1 localization

• Antibody titration: Determine the optimal concentration that maximizes specific signal while minimizing background

• Alternative antibodies: Compare results using antibodies targeting different NF1 epitopes

• Genetic models: Validate signals using NF1 knockout/knockdown models or known NF1 mutation models

• Blocking peptides: Pre-incubate antibody with immunizing peptide to confirm signal specificity

• Application-specific controls: For flow cytometry, use unlabelled samples and isotype controls as demonstrated in search result

How should I interpret variations in NF1 protein levels across different experimental contexts?

Variations in NF1 detection require careful interpretation considering multiple factors:

• Mutation effects: Different NF1 mutations can affect protein stability, expression, and function differently. The Y489C variant creates a new splice donor site resulting in a frameshift and premature stop codon, abolishing NF1 protein function

• Tissue-specific regulation: NF1 expression varies across tissue types, as evident from the differential staining patterns in various cancer tissues

• Developmental context: NF1 expression and function may change during development

• Disease state influence: Pathological conditions may alter NF1 levels independent of genetic mutations

• Signaling feedback loops: Ras pathway activation status can influence NF1 expression through feedback mechanisms

• Technical variables: Differences in sample preparation, antibody lots, or detection methods can affect quantitative measurements

• Treatment effects: Therapeutic interventions, such as PMO treatment, can restore protein levels to approximately 30% of wild-type levels, which may be sufficient for functional restoration

• Splice variants: Alternative splicing generates multiple NF1 isoforms that may be detected differently by various antibodies

How can I optimize NF1 detection in challenging sample types?

Detecting NF1 in difficult samples requires specialized approaches:

• For tissues with high background:

  • Increase blocking stringency (use 10% goat serum as shown in IHC protocols)

  • Optimize primary antibody concentration (2 μg/ml for IHC applications)

  • Extend washing steps to reduce non-specific binding

• For samples with low NF1 expression:

  • Increase protein loading (50 μg for Western blot)

  • Use sensitive detection methods (enhanced chemiluminescence)

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

• For fixed tissues with potential epitope masking:

  • Optimize antigen retrieval (EDTA buffer, pH 8.0)

  • Try multiple antibodies targeting different epitopes

  • Consider alternative fixation methods if possible

• For degradation-prone samples:

  • Process samples rapidly at cold temperatures

  • Use comprehensive protease inhibitor cocktails

  • Avoid repeated freeze-thaw cycles

• For samples with interfering substances:

  • Perform additional purification steps before analysis

  • Try alternative extraction buffers

  • Consider immunoprecipitation before Western blot to concentrate the target protein