NOTCH2 Antibody, HRP conjugated

What is the optimal protocol for Western blot analysis using NOTCH2 antibody with HRP conjugation?

For optimal Western blot results with HRP-conjugated NOTCH2 antibodies, follow this validated protocol:

• Prepare cell/tissue lysates in standard RIPA buffer supplemented with protease inhibitors

• Load 10-30 μg of protein per lane on 8-10% SDS-PAGE gels (NOTCH2 has high molecular weight forms)

• Transfer to PVDF membrane (recommended over nitrocellulose for better protein retention)

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

• Incubate with HRP-conjugated NOTCH2 antibody at 1:1000-1:4000 dilution overnight at 4°C

• Wash 3-4 times with TBST

• Develop using chemiluminescence detection system

Expected bands: Full-length NOTCH2 at ~265 kDa and cleaved forms at ~110 kDa and ~72 kDa (cleaved N-terminus)

Note: Freshly prepared lysates yield better results than frozen samples, particularly for detecting cleaved forms .

How do different NOTCH2 antibodies compare in their ability to detect specific forms of the protein?

NOTCH2 antibodies vary significantly in their epitope recognition and detection capabilities:

The choice depends on research objectives: cleaved N-terminus antibodies are ideal for studying activation, while intracellular domain antibodies better detect nuclear signaling events .

What are the most reliable validation methods to confirm NOTCH2 antibody specificity?

Comprehensive validation of NOTCH2 antibody specificity should include:

• Dot blot analysis against recombinant NOTCH family proteins (NOTCH1, NOTCH2, NOTCH3) to confirm isoform specificity

• Genetic validation using NOTCH2 knockout/knockdown cells as negative controls

• Peptide competition assay with the immunizing peptide

• Orthogonal validation comparing results from antibodies targeting different NOTCH2 epitopes

• Signal induction testing showing increased signal following γ-secretase cleavage activation

• Cross-species reactivity testing if working with non-human models

Additionally, independent validation from established labs provides strong evidence of antibody reliability, such as validation performed at the University of Ulm for certain NOTCH2 antibodies .

What are the critical considerations when using HRP-conjugated NOTCH2 antibodies for immunohistochemistry?

For successful IHC with HRP-conjugated NOTCH2 antibodies:

• Epitope retrieval is crucial: Heat-induced epitope retrieval using citrate buffer (pH 6.0) or basic retrieval reagents (pH 9.0) is essential, as demonstrated in validated protocols

• Fixation impacts detection: For cleaved NOTCH2 forms, use 4% paraformaldehyde with shorter fixation times

• Optimal dilution range: 1:50-1:500 depending on specific antibody and tissue type

• Signal amplification: For low-abundance detection, consider tyramide signal amplification systems

• Nuclear vs. membrane staining interpretation: Cleaved forms show nuclear localization while full-length forms show membrane/cytoplasmic staining

Example protocol: Subject tissue to heat-induced epitope retrieval, incubate with primary NOTCH2 antibody (5 μg/ml) for 1 hour at room temperature, followed by HRP-polymer secondary antibody, and develop with DAB .

How does NOTCH2 antibody performance vary across different species and tissue types?

NOTCH2 antibody performance shows significant species and tissue-dependent variation:

For pancreatic tissues, antibodies show differential staining patterns across developmental stages (day 0 to day 14), with stronger detection in pancreatic progenitor cells compared to pluripotent stem cells .

For bone tissue research, NOTCH2 antibodies have been validated in models of Hajdu-Cheney syndrome, demonstrating efficacy in detecting pathological changes in osseous tissues .

How can researchers optimize protocols for detecting NOTCH2 activation in signaling pathway studies?

To effectively study NOTCH2 activation in signaling pathways:

• Use complementary antibodies: Combine antibodies recognizing both full-length and cleaved forms to track processing

• Time-course experiments: NOTCH2 cleavage occurs sequentially (S1→S2→S3); sample collection timing is critical

• Subcellular fractionation: Separate nuclear and membrane fractions to track NICD translocation

• Co-immunoprecipitation: Use NOTCH2 antibodies to pull down interaction partners (RBPJ/RBPSUH)

• Inhibitor controls: Include γ-secretase inhibitors as negative controls

For optimal results tracking NOTCH2 activation in B cells, samples should be collected at specific timepoints after immunization (days 7 and 14) when NOTCH2 signaling regulates fate decisions between germinal center and marginal zone B cells .

What are the advanced applications of NOTCH2 antibodies in therapeutic research models?

NOTCH2 antibodies have demonstrated significant therapeutic potential in research models:

• Reversing bone phenotypes: Anti-NOTCH2 NRR antibody (10 mg/kg twice weekly for 4 weeks) successfully reversed osteopenic phenotype in Hajdu-Cheney syndrome mouse models by locking the receptor in its quiescent state

• Modulating B cell differentiation: NOTCH2 antibodies can manipulate the fate decision between germinal center and marginal zone B cell development, with potential applications in immunomodulation

• Combination therapies: When used alongside inhibitors of other pathways, NOTCH2 antibodies show enhanced efficacy

Key dose considerations: The effective dose range of 5-10 mg/kg for in vivo applications balances efficacy with minimal gastrointestinal toxicity, based on immunoglobulin G half-life data .

How should researchers interpret conflicting results between different NOTCH2 antibody detection methods?

When facing contradictory NOTCH2 antibody results:

• Consider epitope accessibility: The NOTCH2 epitope recognized by certain antibodies is only exposed after γ-secretase cleavage and remains inaccessible in uncleaved forms

• Validate processing state: NOTCH2 undergoes multiple cleavage events (S1, S2, S3) producing fragments of different sizes:

  • S1 cleavage by furin-like convertase in trans-Golgi

  • S2 cleavage by TACE upon ligand binding

  • S3 cleavage by γ-secretase releasing NICD

• Cross-check with multiple antibodies: Use antibodies targeting different domains (NRR, NICD) to confirm findings

• Assess experimental conditions: Ligand stimulation status (Jagged1/2, Delta1) dramatically affects detection patterns

• Consider tissue-specific processing: Different tissues show varying NOTCH2 processing kinetics and fragment accumulation

What technical challenges exist when conjugating NOTCH2 antibodies with HRP?

HRP conjugation of NOTCH2 antibodies presents several technical challenges:

• Epitope masking: Direct HRP conjugation may affect binding site accessibility, particularly for antibodies targeting the cleaved form where the epitope region is small and conformationally sensitive

• Optimal conjugation ratio: The recommended HRP:antibody molar ratio is typically 4:1 for NOTCH2 antibodies, but requires optimization based on antibody concentration and target application

• Preserving functionality: Recombinant antibody formats (Superclonal™ antibodies) maintain better functionality after conjugation compared to traditional polyclonal antibodies

• Storage stability: HRP-conjugated NOTCH2 antibodies show reduced shelf-life (3-6 months) compared to unconjugated antibodies (1-2 years)

• Alternative approach: Consider using high-sensitivity unconjugated primary antibodies with HRP-conjugated secondary antibodies for maximum flexibility and sensitivity

How can NOTCH2 antibodies be effectively used in multiparameter analyses?

For complex multiparameter analyses involving NOTCH2:

• Multiplex immunofluorescence:

  • Compatible fluorophore combinations for NOTCH2 co-staining include:

  • NOTCH2 (AF488) + Jagged1 (AF647) + DAPI (nuclear)

  • NOTCH2 (HRP-tyramide-Cy3) + CD3 (AF647) + CD20 (AF488)

• Flow cytometry applications:

  • Optimize fixation/permeabilization for intracellular NOTCH2 detection

  • Consider using hamster monoclonal HMN2-35 antibody specifically validated for FACS

  • Critical controls: FMO (fluorescence minus one) controls are essential

• Single-cell analysis protocols:

  • NOTCH2 antibodies have been validated in ChIP-seq applications

  • For single-cell mass cytometry, metal-conjugated NOTCH2 antibodies provide superior resolution

• Spatial protein profiling:

  • NOTCH2 antibodies compatible with digital spatial profiling technologies enable neighborhood context analysis

  • Validated for co-detection with transcription factors like Irf4 in spatial contexts

What are the most effective negative controls for validating NOTCH2 antibody specificity?

Robust negative controls for NOTCH2 antibody validation include:

• Isotype controls: Matched concentration of non-specific rabbit IgG (for rabbit-derived NOTCH2 antibodies)

• Cell line controls: IMR-32 cells have been validated as negative controls for certain NOTCH2 antibodies

• Developmental stage controls: Human pluripotent stem cells (day 0) and definite endoderm cells (day 4) show minimal NOTCH2 expression compared to pancreatic progenitor cells (day 14)

• Peptide competition: Pre-incubation with immunizing peptide should abolish specific signal

• Genetic models: Notch2 conditional knockout cells using CD19-Cre or Cγ1-Cre systems provide definitive negative controls

• Cross-reactivity panels: Test against related NOTCH family members (NOTCH1, NOTCH3) using dot blot analysis with recombinant protein fragments

How does NOTCH2 antibody selection impact detection of disease-associated mutations and variants?

Selection of appropriate NOTCH2 antibodies is critical for studying disease-related mutations:

• Hajdu-Cheney syndrome (HCS):

  • The Notch2 Q2319X mutation creates a truncated protein

  • Antibodies targeting the N-terminal or middle domains can detect the mutant protein, while C-terminal antibodies cannot

• Alagille syndrome type 2 (ALGS2):

  • Caused by NOTCH2 loss-of-function mutations

  • Requires antibodies specific to functional domains affected by patient-specific mutations

• Cancer-associated NOTCH2 alterations:

  • NOTCH2 amplification in certain cancers requires quantitative detection approaches

  • Antibodies specific to activated forms help distinguish pathological from physiological activation

Research approach recommendation: Use complementary antibodies targeting different domains to comprehensively characterize variant forms and their functional impacts.

What are the key methodological considerations for NOTCH2 antibody use in developmental studies?

For developmental biology applications:

• Temporal expression dynamics:

  • NOTCH2 expression varies significantly across developmental stages

  • Antibody selection should account for stage-specific processing and expression levels

• Tissue fixation protocols:

  • For embryonic tissues: 2% paraformaldehyde for 2-4 hours preserves epitope accessibility

  • For adult tissues: 4% paraformaldehyde overnight may be required

• Validated developmental models:

  • Pancreatic development: NOTCH2 antibodies validated across differentiation from human pluripotent stem cells to pancreatic progenitor cells

  • B cell development: NOTCH2 antibodies track fate decisions between germinal center B cells and marginal zone B cells

• Co-staining markers:

  • Pair NOTCH2 antibodies with lineage-specific markers to track cell fate decisions

  • For B cell development: CD19, CD21, CD23 combined with NOTCH2 provides comprehensive profiling

What approaches should be used when optimizing NOTCH2 antibody dilutions for novel applications?

Systematic optimization of NOTCH2 antibody dilutions should follow this approach:

• Titration matrix:

  • Start with manufacturer's recommended range (e.g., 1:1000-1:4000 for WB, 1:50-1:500 for IHC)

  • Perform systematic titration across at least 5 dilution points

  • Include positive and negative controls at each dilution

• Application-specific considerations:

  • Western blot: Lower dilutions (1:1000) for cleaved forms, higher dilutions (1:4000) for abundant full-length forms

  • IHC: Lower dilutions (1:50) for tissue samples, higher dilutions (1:500) for cell lines with overexpression

  • ELISA: Ultra-high dilutions (1:30,000 to 1:90,000) have been validated for certain antibodies

• Signal-to-noise optimization:

  • Plot signal-to-background ratio at each dilution point

  • Select optimal dilution at highest specificity without signal intensity loss

• Sample-specific adjustments:

  • Increase concentration for fixed tissues where epitope accessibility may be limited

  • Decrease concentration for overexpression systems to prevent signal saturation

How can NOTCH2 antibodies be used to investigate pathway crosstalk in complex biological systems?

For investigating NOTCH2 pathway interactions:

• Validated co-immunoprecipitation protocols:

  • NOTCH2 antibodies can pull down interaction partners including RBPJ/RBPSUH, revealing key pathway components

  • Optimal lysis conditions: 1% NP-40 buffer with low salt (150mM NaCl) preserves interactions

• Multiple pathway activation markers:

  • Combine NOTCH2 antibodies with antibodies against:

    • NFATc1 to study osteoclast differentiation pathways

    • RELA/p65 to investigate NFκB crosstalk

    • Irf4 to examine B cell differentiation programs

• Temporal dynamics analysis:

  • NOTCH2 signaling shows distinct temporal patterns after activation

  • Day 7 vs. day 14 analysis reveals different roles in early vs. late immune responses

• Tissue context considerations:

  • In bone tissue: NOTCH2 antibodies reveal interaction with RANKL-induced pathways

  • In B cells: NOTCH2 antibodies demonstrate crosstalk between antigen-induced activation and Notch signaling

What are the most reliable protocols for using NOTCH2 antibodies in chromatin immunoprecipitation (ChIP) assays?

Optimized ChIP protocol for NOTCH2:

• Crosslinking conditions:

  • 1% formaldehyde for 10 minutes at room temperature

  • Quench with 125mM glycine for 5 minutes

• Chromatin fragmentation:

  • Sonication to achieve fragments of 200-500bp

  • Verify fragmentation efficiency by gel electrophoresis

• Antibody selection:

  • Use antibodies targeting the intracellular domain (NICD) for transcription factor binding detection

  • For ChIP-seq applications, antibodies against NOTCH2 C-terminal region are validated

• Input requirements:

  • 5-10 μg of chromatin per IP reaction

  • 2-5 μg of NOTCH2 antibody per reaction

• Controls:

  • IgG negative control

  • Known NOTCH2 target regions (Hes1/5 promoters) as positive controls

Key quality control: Verify enrichment at canonical NOTCH2 binding sites (RBP-J binding motifs) compared to non-target regions.

What are the current limitations of commercially available NOTCH2 antibodies in research applications?

Current limitations of NOTCH2 antibodies include:

• Isoform specificity challenges:

  • Limited ability to distinguish between closely related NOTCH family members

  • Dot blot analysis shows potential cross-reactivity between NOTCH1, NOTCH2, and NOTCH3

• Processing state detection:

  • Many antibodies cannot simultaneously detect both cleaved and uncleaved forms

  • The immunogen epitope for some antibodies is only exposed after γ-secretase cleavage

• Species cross-reactivity limitations:

  • While sequence homology predicts cross-reactivity with mouse and rat, actual validation data is limited

  • Most robust validation exists for human samples

• Lot-to-lot variability:

  • Traditional polyclonal antibodies show significant batch variation

  • Recombinant antibody technologies (Superclonal™, recombinant monoclonal) offer improved consistency

• Application restrictions:

  • Limited validation for emerging techniques like mass cytometry, CODEX, and spatial transcriptomics

  • Most antibodies are validated primarily for conventional applications (WB, IHC, ELISA)

How do Recombinant Superclonal™ NOTCH2 antibodies compare to traditional monoclonal and polyclonal antibodies?

Comparative analysis of NOTCH2 antibody formats:

Recombinant Superclonal™ antibodies combine advantages of both formats: they recognize multiple epitope sites like polyclonals while maintaining the consistency of monoclonals through a known mixture of light and heavy chains .

What are best practices for long-term storage and handling of NOTCH2 antibodies to maintain functionality?

Optimal storage and handling protocols for NOTCH2 antibodies:

• Storage conditions:

  • Primary storage: -20°C in aliquots to prevent freeze-thaw cycles

  • Working dilutions: 4°C for up to one week

  • Storage buffer: PBS with 0.02% sodium azide and 50% glycerol pH 7.3

• Aliquoting strategy:

  • For 200μL stock, create 10-20μL aliquots to minimize freeze-thaw cycles

  • Small sizes (20μL) containing 0.1% BSA provide additional stability

• Stability considerations:

  • Unconjugated antibodies: Stable for one year after shipment at -20°C

  • HRP-conjugated antibodies: More sensitive to degradation, use within 6 months

• Avoiding performance loss:

  • Minimize exposure to direct light (especially for fluorophore-conjugated antibodies)

  • Centrifuge briefly before opening to collect solution at bottom of vial

  • Add carrier protein (0.1-0.5% BSA) to diluted antibodies for extended storage

• Quality control monitoring:

  • Include positive control samples in each experiment to track performance over time

  • Document lot numbers and maintain consistent sourcing when possible

What are the emerging applications of NOTCH2 antibodies in therapeutic development research?

Cutting-edge therapeutic applications for NOTCH2 antibodies include:

• Targeted inhibition strategies:

  • Anti-NOTCH2 NRR antibodies lock the receptor in its quiescent state, preventing activation

  • Demonstrated efficacy in reversing osteopenia in Hajdu-Cheney syndrome models

  • Dosing protocol: 10 mg/kg twice weekly for 4 weeks shows therapeutic effect with minimal toxicity

• Immunomodulatory approaches:

  • NOTCH2 signaling controls fate decisions between germinal center and marginal zone B cells

  • Manipulating this pathway could tune antibody responses in vaccination or autoimmunity

• Combination therapy research:

  • NOTCH2 antibodies combined with inhibitors of complementary pathways show enhanced efficacy

  • Integration with JAG1/JAG2 targeting approaches provides multi-level pathway control

• Cancer research applications:

  • NOTCH2 positively regulates self-renewal of liver cancer cells

  • Antibodies detecting activated forms help distinguish pathological from physiological activation

• Safety profile research:

  • Information on antibody-dependent gastrointestinal toxicity shows acceptable safety profile at doses of 5 mg/kg

  • Half-life studies based on immunoglobulin G pharmacokinetics inform dosing schedules