Cleaved-NOTCH1 (V1754) Antibody

What is Cleaved-NOTCH1 (V1754) Antibody and what epitope does it specifically recognize?

The Cleaved-NOTCH1 (V1754) Antibody is a polyclonal antibody raised in rabbits that specifically detects endogenous levels of the cytosolic domain of Notch1 protein only when cleaved between Gly1743 and Val1744 . This antibody recognizes the active form of the Notch1 protein resulting from proteolytic processing but does not recognize full-length Notch1 or Notch1 cleaved at other positions . The immunogen is typically a synthesized peptide derived from the internal region of human Notch1, specifically within the amino acid range 1735-1784 .

The specific detection of this cleaved form is crucial for studying Notch signaling activation, as the cleaved intracellular domain (NICD) translocates to the nucleus where it functions as a transcriptional regulator .

What are the validated applications for Cleaved-NOTCH1 (V1754) Antibody?

The Cleaved-NOTCH1 (V1754) Antibody has been validated for multiple research applications:

These applications have been validated across multiple manufacturers including antibodies with catalog numbers orb675227, E-AB-30054, and STJ90066 .

Why is there a discrepancy between calculated and observed molecular weight in Western blot?

The calculated molecular weight of full-length Notch1 is approximately 273 kDa, while the observed molecular weight of the cleaved Notch1 intracellular domain is typically around 110 kDa . This discrepancy occurs because:

• The antibody specifically detects only the cleaved intracellular domain (NICD) of Notch1, not the full-length protein

• Post-translational modifications may affect protein mobility in gels

• Proteolytic processing results in a fragment that is significantly smaller than the full-length protein

As noted in the technical documentation: "The mobility is affected by many factors, which may cause the observed band size to be inconsistent with the expected size. If a protein in a sample has different modified forms at the same time, multiple bands may be detected on the membrane."

What are the optimal storage and handling conditions for preserving antibody activity?

For optimal antibody performance, the following storage conditions are recommended:

• Short-term storage (up to 2 weeks): Maintain refrigerated at 2-8°C

• Long-term storage (up to 12 months): Store at -20°C in small aliquots

• Avoid repeated freeze-thaw cycles to maintain antibody integrity

• The antibody is typically supplied in PBS containing 50% glycerol, 0.5% BSA/rAlbumin, and 0.02% sodium azide as preservatives

Upon receipt, it is recommended to immediately store the antibody at the recommended temperature .

How can I quantitatively assess differences in cleaved Notch1 activity between normal and tumor tissues?

To quantitatively assess cleaved Notch1 activity differences between normal and tumor tissues, ELISA is the recommended approach. Based on published protocols:

• Collect matched normal and tumor tissue samples

• Lyse tissues and quantitate protein levels using a protein assay kit (e.g., Pierce Protein Assay)

• Plate equal amounts of protein on the ELISA plate designed to detect cleaved Notch1 (Val1744)

• Incubate for 2 hours following manufacturer's protocol

• Read absorbance at 450 nm using a plate reader

• Compare absorbance values between normal and tumor samples using statistical analysis (paired Student's t-test)

This methodology has been successfully employed to demonstrate differences in cleaved Notch1 activity between normal colon and colorectal cancer tissues . Researchers should consider biological replicates (n≥9) to ensure statistical significance and include appropriate controls.

Statistical significance should be determined with p-values < 0.05 considered significant .

What experimental controls should be included when using Cleaved-NOTCH1 (V1754) Antibody for Western blot analysis?

When conducting Western blot analysis with Cleaved-NOTCH1 (V1754) Antibody, the following controls should be included:

• Positive controls:

  • Mouse brain tissue lysates (verified sample)

  • Rat muscle tissue lysates (verified sample)

  • KB cell lysates (validated cell line)

  • HEK 293-A cells (though band size may be slightly off from expected)

• Negative controls:

  • Lysates from cells with NOTCH1 knockdown (siRNA)

  • Samples treated with gamma-secretase inhibitors (prevents Notch1 cleavage)

  • Isotype control antibody

• Loading controls:

  • Housekeeping proteins (β-actin, GAPDH, etc.)

  • Total protein stain (for normalization)

• Technical considerations:

  • Use 40 μg of protein resolved over 4-20% Tris-glycine gels

  • Transfer onto nitrocellulose membranes

  • Proper dilution of primary antibody (1:500-1:2000)

  • Perform densitometric measurements of bands using appropriate software

Proper controls ensure specificity of detection and allow for meaningful interpretation of results.

How can I design experiments to investigate the role of cleaved Notch1 in cancer invasion and metastasis?

Based on published research approaches, the following experimental design can be employed to investigate cleaved Notch1's role in cancer invasion and metastasis:

• Expression analysis:

  • Compare cleaved Notch1 levels between normal and tumor tissues using Western blot and ELISA

  • Correlate with clinical outcomes (survival, metastasis) in patient cohorts

• Functional studies:

  • Targeted knockdown of Notch1 using siRNA technology in cancer cell lines

  • Assess the effect on invasion using in vitro invasion assays

  • Monitor expression of invasion-related genes including MMPs, uPA, and uPAR by Western blot

• Gene expression profiling:

  • Use pathway-focused arrays (e.g., Human Extracellular Matrix and Adhesion molecules oligo gene array)

  • Compare gene expression profiles between control cells and Notch1-knockdown cells

  • Analyze data using appropriate software (e.g., GEArray Expression Analysis Suite)

• In vivo studies:

  • Develop patient-derived tumor xenograft (PDTX) models

  • Compare tumors with and without NOTCH1 gene copy number gain

  • Test sensitivity to Notch1-targeting antibodies

  • Monitor tumor growth, invasion, and metastasis

This comprehensive approach has been validated in prostate cancer and colorectal cancer studies, where Notch1 knockdown inhibited cancer cell invasion by modulating the expression of invasion-related genes .

What is the relationship between NOTCH1 gene copy number and cleaved Notch1 protein expression?

Research has established a significant relationship between NOTCH1 gene copy number and cleaved Notch1 protein expression, particularly in colorectal cancer:

• Gene copy number assessment:

  • NOTCH1 gene copy number gain has been identified in a subset of colorectal cancer (CRC) patients

  • This genomic alteration is associated with worse survival in patients with advanced CRC

• Protein expression correlation:

  • Tumors harboring a NOTCH1 gain exhibit significant elevation of:

    • Notch1 receptor expression

    • JAG1 ligand expression

    • Cleaved Notch1 activity

• Therapeutic implications:

  • A significant association exists between NOTCH1 gene copy number gain and sensitivity to Notch1-targeting antibodies

  • This suggests potential for targeted therapy in patients with NOTCH1 amplification

• Experimental approach:

  • Compare cleaved Notch1 levels between tumors with and without NOTCH1 gene amplification

  • Use patient-derived tumor xenograft (PDTX) models for functional validation

  • Test response to Notch1-targeting therapeutics

This relationship provides a mechanistic basis for the poor prognosis observed in patients with NOTCH1 gene copy number gain and identifies a potential biomarker for selecting patients who might benefit from Notch1-targeted therapies .

How should I optimize immunohistochemistry protocols for Cleaved-NOTCH1 (V1754) Antibody?

For optimal immunohistochemistry results with Cleaved-NOTCH1 (V1754) Antibody:

• Tissue preparation:

  • Use formalin-fixed, paraffin-embedded (FFPE) tissue sections

  • Perform antigen retrieval (heat-induced epitope retrieval in citrate buffer pH 6.0 is typically recommended)

  • Block endogenous peroxidase activity with hydrogen peroxide

• Antibody application:

  • Optimal dilution range: 1:50-1:300 for IHC-P applications

  • Start with 1:100 dilution and optimize based on signal intensity and background

  • Incubate at 4°C overnight for best results

• Detection system:

  • Use appropriate secondary antibody conjugated to HRP

  • Develop with DAB substrate

  • Counterstain with hematoxylin

• Validated positive controls:

  • Rat brain tissue has been verified as a positive control

  • Consider including tissue samples known to express high levels of cleaved Notch1 (e.g., certain cancer tissues)

• Negative controls:

  • Omit primary antibody

  • Use isotype control antibody

  • Include tissues known to express low/no cleaved Notch1

• Quantification:

  • Use digital image analysis software for quantification

  • Score based on staining intensity and percentage of positive cells

  • Compare with other methods (WB, ELISA) for validation

Optimization may require adjusting antibody concentration, incubation time, and antigen retrieval conditions based on specific tissue types and experimental goals.

How can I validate the specificity of Cleaved-NOTCH1 (V1754) Antibody in my experimental system?

To validate antibody specificity in your experimental system:

• Multiple detection methods:

  • Compare results from Western blot, IHC, IF, and ELISA using the same samples

  • Consistent patterns across methods increase confidence in specificity

• Positive and negative controls:

  • Use verified positive control samples: Mouse brain, rat muscle, KB cells

  • Include samples with Notch1 knockdown (siRNA treatment)

  • Use gamma-secretase inhibitors to block Notch1 cleavage as negative controls

• Band size verification:

  • Confirm that the detected band is approximately 110 kDa, corresponding to cleaved Notch1

  • Be aware that the observed molecular weight may differ from calculated values due to post-translational modifications

• Functional validation:

  • Correlate cleaved Notch1 detection with downstream Notch signaling activation

  • Assess nuclear localization of the detected protein (cleaved Notch1 translocates to the nucleus)

• Antibody validation panel:

  • Test multiple antibodies against different epitopes of Notch1

  • Compare antibodies from different manufacturers or clones

These approaches collectively strengthen confidence in antibody specificity and reliability of experimental results.

What are the best practices for quantifying Western blot results when using Cleaved-NOTCH1 (V1754) Antibody?

For accurate quantification of Western blot results:

• Sample preparation:

  • Use 40 μg of protein per lane as a starting point

  • Ensure equal loading across all lanes using total protein normalization

  • Include gradient of known concentrations for standard curve generation

• Electrophoresis and transfer:

  • Use 4-20% Tris-glycine gels for optimal separation

  • Ensure complete and uniform transfer to nitrocellulose membranes

  • Verify transfer efficiency with reversible staining

• Antibody incubation:

  • Use optimized antibody dilution (1:500-1:2000)

  • Ensure consistent incubation times and conditions between experiments

• Detection and imaging:

  • Use digital imaging systems within linear dynamic range

  • Avoid overexposure which prevents accurate quantification

  • Capture multiple exposures to ensure linearity

• Densitometric analysis:

  • Use validated scientific software (e.g., UN-SCAN-IT)

  • Subtract background appropriately and consistently

  • Normalize to loading controls or total protein

• Statistical analysis:

  • Perform experiments in triplicate (minimum)

  • Apply appropriate statistical tests based on experimental design

  • Report quantification with error bars and statistical significance

Following these practices ensures reliable quantification of cleaved Notch1 levels and facilitates comparison across different experimental conditions or samples.

What are common issues encountered when using Cleaved-NOTCH1 (V1754) Antibody and how can they be resolved?

Note: The Cleaved-NOTCH1 (V1754) Antibody specifically detects the cleaved form at Val1754 and does not recognize full-length Notch1, which may explain absence of the 273 kDa band .

How can I improve signal detection in tissues with low levels of cleaved Notch1?

For improving detection in low-expressing samples:

• Sample preparation optimization:

  • Enrich for nuclear fraction (cleaved Notch1 translocates to nucleus)

  • Use phosphatase inhibitors to preserve modification status

  • Consider using cell/tissue types known to have active Notch signaling

• Signal amplification methods:

  • Employ tyramide signal amplification (TSA) for IHC/IF

  • Use more sensitive detection systems (chemiluminescent vs. colorimetric)

  • Consider using high-sensitivity ECL substrates for Western blot

• Protocol adjustments:

  • Increase antibody concentration (use 1:50 dilution for IHC/IF)

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

  • Optimize antigen retrieval conditions

• Alternative detection methods:

  • ELISA may provide greater sensitivity for quantitative analysis

  • Consider using more sensitive detection techniques like droplet digital PCR for gene expression

• Notch pathway modulation:

  • Consider treatment with Notch ligands (Jagged, Delta) to increase cleaved Notch1 levels

  • Inhibit protein degradation pathways to increase protein stability

These approaches can significantly improve detection sensitivity while maintaining specificity for the cleaved form of Notch1.

How can Cleaved-NOTCH1 (V1754) Antibody be used in cancer research?

The Cleaved-NOTCH1 (V1754) Antibody has multiple applications in cancer research:

• Biomarker identification:

  • Detection of cleaved Notch1 levels in patient samples can serve as a prognostic biomarker

  • NOTCH1 gene copy number gain is associated with worse survival in colorectal cancer patients

  • Elevated cleaved Notch1 can identify tumors with active Notch signaling

• Therapeutic target assessment:

  • Evaluate response to Notch1-targeting antibodies in tumors with NOTCH1 gain

  • Monitor Notch1 activation status before and after treatment

  • Identify patient populations likely to respond to Notch pathway inhibitors

• Invasion and metastasis studies:

  • Knockdown of Notch1 inhibits invasion of human prostate cancer cells

  • Monitor expression of invasion-related genes modulated by Notch1

  • Study the relationship between Notch1 activation and metastatic potential

• Patient-derived models:

  • Characterize Notch1 activation in patient-derived tumor xenograft (PDTX) models

  • Compare normal vs. tumor tissue from the same patient

  • Correlate cleaved Notch1 levels with patient outcomes

• Combination with other markers:

  • Co-staining with other cancer biomarkers

  • Multiplex analysis to understand pathway interactions

  • Correlation with other components of the Notch signaling pathway (JAG1 ligand)

These applications provide valuable insights into cancer biology and potential therapeutic strategies targeting the Notch signaling pathway.

What is the role of cleaved Notch1 in development and how can this antibody be used in developmental biology research?

Cleaved Notch1 plays critical roles in development, and the Cleaved-NOTCH1 (V1754) Antibody can be valuable for developmental biology research:

• Developmental functions of Notch1:

  • Cell fate determination during embryonic development

  • Neurogenesis and neural development (verified in rat brain samples)

  • Vascular development and angiogenesis

  • Stem cell maintenance and differentiation

• Experimental applications:

  • Temporal tracking of Notch1 activation during developmental stages

  • Spatial mapping of cleaved Notch1 in developing tissues using IHC/IF

  • Quantitative assessment of Notch1 activity in different cell populations

• Stem cell research:

  • Monitor Notch1 activation during stem cell differentiation

  • Compare cleaved Notch1 levels between stem cells and differentiated progeny

  • Study the effect of Notch pathway modulation on stem cell fate

• Organ development studies:

  • Track Notch1 activation in organogenesis

  • Correlate Notch signaling with tissue patterning and morphogenesis

  • Study the effect of Notch1 inhibition on developmental processes

• Disease modeling:

  • Investigate developmental abnormalities associated with Notch pathway dysregulation

  • Study congenital disorders linked to Notch signaling defects

  • Model developmental aspects of cancer initiation

The antibody's validation in mouse brain and rat brain tissues makes it particularly suitable for neurodevelopmental studies , while its application in multiple species (human, mouse, rat) enables comparative developmental research.