NBN (Ab-343) Antibody

What is NBN (Ab-343) Antibody and what epitope does it recognize?

NBN (Ab-343) Antibody is a polyclonal antibody produced in rabbit that specifically recognizes the peptide sequence around amino acids 341-345 (S-L-S-Q-G) of human p95/NBS1 protein. It detects endogenous levels of total NBN protein regardless of phosphorylation status . This antibody has been generated by immunizing rabbits with a synthetic peptide-KLH conjugate corresponding to this sequence region and subsequently purified via affinity chromatography using epitope-specific peptide .

NBN is a crucial component of the MRE11/RAD50/NBN (MRN) complex involved in DNA double-strand break repair and DNA damage-induced checkpoint activation. The region around amino acid 343 is particularly important as it contains a key ATM phosphorylation site (Ser343) that regulates NBN function in DNA damage response .

What validated applications can NBN (Ab-343) Antibody be used for?

The NBN (Ab-343) Antibody has been validated for multiple experimental applications:

Validation data shows specific detection in Jurkat cell lysates and human breast carcinoma tissue, with specificity confirmed through peptide competition assays . For Western blot applications, researchers typically observe a band at approximately 95 kDa, consistent with the expected molecular weight of full-length NBN protein .

What is the biological significance of NBN protein in cellular physiology?

NBN (Nibrin) plays several critical roles in maintaining genomic integrity:

• DNA double-strand break (DSB) repair: As part of the MRN complex, NBN helps detect and initiate repair of DSBs

• Cell cycle checkpoint activation: Phosphorylation of NBN by ATM is required for S-phase checkpoint activation

• MRN complex formation: NBN facilitates the proper assembly and localization of the MRE11/RAD50/NBN complex

• Genomic stability maintenance: Proper NBN function prevents chromosomal aberrations

Mutations in the NBN gene are associated with Nijmegen breakage syndrome, an autosomal recessive disorder characterized by microcephaly, growth retardation, immunodeficiency, and cancer predisposition . The protein is thought to be involved in DNA damage-induced checkpoint activation, making it a crucial component of cellular responses to genotoxic stress .

Research by Larsen et al. demonstrated that phosphorylation of NBN regulates its accumulation at DNA break sites, as well as the accumulation of ATM, influencing both the timing and efficiency of DNA repair processes .

How should NBN (Ab-343) Antibody be stored and handled for optimal performance?

For optimal performance and longevity of the NBN (Ab-343) Antibody:

Critical handling guidelines:

• Avoid repeated freeze-thaw cycles as they can denature the antibody and reduce effectiveness

• Aliquot the stock solution upon first thaw to minimize freeze-thaw cycles

• Use sterile technique when handling to prevent contamination

• Keep cold during handling and return to appropriate storage temperature promptly

• For diluted working solutions, prepare fresh when possible or store according to buffer stability

What is the relationship between NBN (Ab-343) Antibody and phospho-specific NBN antibodies?

NBN (Ab-343) Antibody recognizes total NBN protein regardless of phosphorylation status, while phospho-specific antibodies only detect NBN when phosphorylated at specific residues:

Research significance:

• Ser343 is a key ATM phosphorylation site critical for S-phase checkpoint activation after DNA damage

• Phosphorylation at this site influences NBN accumulation at DNA break sites and subsequent repair kinetics

• Using both antibody types allows discrimination between changes in total protein levels versus altered phosphorylation states

For comprehensive studies, researchers typically use both antibody types on parallel samples or sequential probing of the same membrane after stripping to calculate the phosphorylation ratio (phospho-NBN/total NBN) .

How can NBN (Ab-343) Antibody be used to track NBN dynamics following DNA damage?

NBN (Ab-343) Antibody is valuable for monitoring NBN dynamics following DNA damage through multiple experimental approaches:

Immunoblotting:

• Perform time-course experiments after DNA damage induction (e.g., 0, 15, 30, 60 min, 2, 4, 8, 24 h)

• Track changes in NBN levels and compare with phosphorylated forms

• Monitor potential proteolytic processing or degradation

Immunofluorescence:

• Visualize redistribution of NBN to nuclear foci at sites of DNA damage

• Co-stain with γH2AX to confirm localization to DNA break sites

• Quantify foci formation kinetics over time

Chromatin Immunoprecipitation (ChIP):
Research by Larsen et al. utilized ChIP with NBN antibodies to track recruitment patterns at site-specific DNA breaks:

• NBN accumulates directly at DNA break sites within 1 hour after damage induction

• Wild-type NBN peaks at 3 hours and then decreases by 5 hours

• Phospho-deficient mutants (S>A) show delayed recruitment, peaking at 5 hours

• Phospho-mimetic mutants (S>E) show prolonged accumulation up to 8 hours

This approach revealed that NBN phosphorylation regulates not only its own recruitment dynamics but also the recruitment of ATM to DNA damage sites, with important implications for DNA repair efficiency .

What controls should be included in experiments using NBN (Ab-343) Antibody?

Proper controls are essential for interpreting results with NBN (Ab-343) Antibody:

Positive Controls:

• Cell lines with known NBN expression (e.g., Jurkat, HeLa)

• Recombinant NBN protein (if available)

• Cells treated with DNA damaging agents (higher phosphorylation)

Negative Controls:

• Antibody pre-incubated with immunizing peptide (blocking peptide control)

• NBN-depleted samples (siRNA knockdown or CRISPR knockout)

• Secondary antibody only

Experimental Controls:

• For DNA damage studies: Paired untreated vs. treated samples

• For phosphorylation studies: Include λ-phosphatase-treated samples

• For cell cycle studies: Synchronized cell populations

Example from Validation Data:
Immunohistochemical analysis of human breast carcinoma tissue shows specific staining when using NBN (Ab-343) Antibody alone, while this staining is abolished when the antibody is pre-incubated with blocking peptide, confirming antibody specificity .

Including these controls ensures confidence in experimental results and facilitates troubleshooting if unexpected results occur.

What methodological approaches enable quantification of NBN phosphorylation kinetics?

To quantify NBN phosphorylation kinetics with precision:

Dual Antibody Approach:

• Use NBN (Ab-343) Antibody to measure total NBN levels

• Use phospho-specific antibodies (e.g., anti-p95/NBS1 phospho S343) for phosphorylated forms

• Calculate phosphorylation ratio (phospho-NBN/total NBN) to normalize for expression differences

Time-course Experimental Design:

Advanced Techniques for Phosphorylation Analysis:

• Phos-tag SDS-PAGE: Separates phosphorylated from non-phosphorylated forms based on mobility shift

• Mass spectrometry: Identifies multiple phosphorylation sites simultaneously and their relative abundances

• FRET-based sensors: Monitors phosphorylation in real-time in living cells

Data Analysis and Interpretation:

• Plot phosphorylation ratio over time to generate kinetic curves

• Calculate rate constants for phosphorylation and dephosphorylation

• Compare kinetics between wild-type and mutant systems

• Correlate with functional outcomes (e.g., DNA repair efficiency, checkpoint activation)

Research by Larsen et al. revealed that phosphorylation-mimicking mutations (S>E) resulted in both increased and prolonged accumulation of NBN at DNA breaks compared to wild-type, while phosphorylation-blocking mutations (S>A) delayed recruitment, demonstrating the regulatory role of phosphorylation in DNA damage response dynamics .

How can chromatin immunoprecipitation (ChIP) with NBN (Ab-343) Antibody be optimized for DNA damage studies?

Optimizing ChIP with NBN (Ab-343) Antibody for DNA damage studies requires careful experimental design:

Protocol Optimization for Site-Specific DNA Breaks:

• Induce site-specific DNA breaks using endonucleases like I-PpoI

• Fix cells with formaldehyde at multiple time points post-damage

• Sonicate chromatin to 200-500 bp fragments

• Immunoprecipitate with 2-5 μg NBN (Ab-343) Antibody

• Design primers for qPCR at various distances from break sites:

  • Directly at break site

  • 1-3 kb from break site

  • 5-10 kb from break site

  • 20+ kb from break site as background control

Key Parameters from Research Literature:

• NBN accumulates directly at DNA break sites, while ATM is detected in regions 3-10 kb flanking the site

• In wild-type cells, NBN is detected at the cleavage site within 1 hour after damage, peaks at 3 hours, and decreases by 5 hours

• At later time points, NBN accumulation shifts to sites 3-10 kb flanking the DSB

• Phosphorylation status significantly impacts both timing and extent of NBN accumulation

Controls and Validation:

• Input chromatin (5-10% of starting material)

• IgG immunoprecipitation as negative control

• Known positive loci (e.g., previously characterized DSB sites)

• Parallel ChIP with antibodies against other DNA damage response proteins (γH2AX, 53BP1)

Larsen et al. demonstrated that blocking NBN phosphorylation (S>A mutations) delayed recruitment to break sites, while phospho-mimetic mutations (S>E) caused prolonged accumulation, revealing the regulatory role of phosphorylation in the spatial and temporal dynamics of the DNA damage response .

How can NBN (Ab-343) Antibody be used in studies comparing DNA repair efficiency between cell lines?

NBN (Ab-343) Antibody can be integrated into comprehensive DNA repair efficiency studies:

Experimental Design for Comparative Repair Studies:

• Select cell lines of interest (e.g., normal vs. cancer, wildtype vs. mutant)

• Induce DNA damage (site-specific or global)

• Monitor repair kinetics over time (0, 1, 3, 5, 8, 24 hours)

• Use multiple readouts to comprehensively assess repair efficiency

Multi-parameter Analysis Workflow:

Insights from Research Literature:

• Larsen et al. found that cells lacking full-length NBN showed significantly delayed repair compared to control cells

• Blocking phosphorylation of NBN resulted in a more modest delay in repair

• Phosphorylation status affected both the timing of recruitment and the duration of NBN presence at break sites

• These findings establish NBN phosphorylation as a regulatory mechanism for DNA repair efficiency

By combining NBN (Ab-343) Antibody with these analytical approaches, researchers can generate comprehensive profiles of DNA repair capabilities across different cell types and genetic backgrounds, providing insights into repair deficiencies associated with cancer or other genomic instability syndromes.

NBN (Ab-343) Antibody: Comprehensive Research FAQs

NBN (Ab-343) Antibody is a valuable research tool for studying DNA damage response pathways, specifically targeting the Nijmegen breakage syndrome protein (NBN/NBS1). This document addresses key research questions from basic protocol optimization to advanced experimental applications based on peer-reviewed literature and technical resources.

What is NBN (Ab-343) Antibody and what epitope does it recognize?

NBN (Ab-343) Antibody is a polyclonal antibody produced in rabbit that specifically recognizes the peptide sequence around amino acids 341-345 (S-L-S-Q-G) of human p95/NBS1 protein. It detects endogenous levels of total NBN protein regardless of phosphorylation status . This antibody has been generated by immunizing rabbits with a synthetic peptide-KLH conjugate corresponding to this sequence region and subsequently purified via affinity chromatography using epitope-specific peptide .

NBN is a crucial component of the MRE11/RAD50/NBN (MRN) complex involved in DNA double-strand break repair and DNA damage-induced checkpoint activation. The region around amino acid 343 is particularly important as it contains a key ATM phosphorylation site (Ser343) that regulates NBN function in DNA damage response .

What validated applications can NBN (Ab-343) Antibody be used for?

The NBN (Ab-343) Antibody has been validated for multiple experimental applications:

Validation data shows specific detection in Jurkat cell lysates and human breast carcinoma tissue, with specificity confirmed through peptide competition assays . For Western blot applications, researchers typically observe a band at approximately 95 kDa, consistent with the expected molecular weight of full-length NBN protein .

What is the biological significance of NBN protein in cellular physiology?

NBN (Nibrin) plays several critical roles in maintaining genomic integrity:

• DNA double-strand break (DSB) repair: As part of the MRN complex, NBN helps detect and initiate repair of DSBs

• Cell cycle checkpoint activation: Phosphorylation of NBN by ATM is required for S-phase checkpoint activation

• MRN complex formation: NBN facilitates the proper assembly and localization of the MRE11/RAD50/NBN complex

• Genomic stability maintenance: Proper NBN function prevents chromosomal aberrations

Mutations in the NBN gene are associated with Nijmegen breakage syndrome, an autosomal recessive disorder characterized by microcephaly, growth retardation, immunodeficiency, and cancer predisposition . The protein is thought to be involved in DNA damage-induced checkpoint activation, making it a crucial component of cellular responses to genotoxic stress .

Research by Larsen et al. demonstrated that phosphorylation of NBN regulates its accumulation at DNA break sites, as well as the accumulation of ATM, influencing both the timing and efficiency of DNA repair processes .

How should NBN (Ab-343) Antibody be stored and handled for optimal performance?

For optimal performance and longevity of the NBN (Ab-343) Antibody:

Critical handling guidelines:

• Avoid repeated freeze-thaw cycles as they can denature the antibody and reduce effectiveness

• Aliquot the stock solution upon first thaw to minimize freeze-thaw cycles

• Use sterile technique when handling to prevent contamination

• Keep cold during handling and return to appropriate storage temperature promptly

• For diluted working solutions, prepare fresh when possible or store according to buffer stability

What is the relationship between NBN (Ab-343) Antibody and phospho-specific NBN antibodies?

NBN (Ab-343) Antibody recognizes total NBN protein regardless of phosphorylation status, while phospho-specific antibodies only detect NBN when phosphorylated at specific residues:

Research significance:

• Ser343 is a key ATM phosphorylation site critical for S-phase checkpoint activation after DNA damage

• Phosphorylation at this site influences NBN accumulation at DNA break sites and subsequent repair kinetics

• Using both antibody types allows discrimination between changes in total protein levels versus altered phosphorylation states

For comprehensive studies, researchers typically use both antibody types on parallel samples or sequential probing of the same membrane after stripping to calculate the phosphorylation ratio (phospho-NBN/total NBN) .

How can NBN (Ab-343) Antibody be used to track NBN dynamics following DNA damage?

NBN (Ab-343) Antibody is valuable for monitoring NBN dynamics following DNA damage through multiple experimental approaches:

Immunoblotting:

• Perform time-course experiments after DNA damage induction (e.g., 0, 15, 30, 60 min, 2, 4, 8, 24 h)

• Track changes in NBN levels and compare with phosphorylated forms

• Monitor potential proteolytic processing or degradation

Immunofluorescence:

• Visualize redistribution of NBN to nuclear foci at sites of DNA damage

• Co-stain with γH2AX to confirm localization to DNA break sites

• Quantify foci formation kinetics over time

Chromatin Immunoprecipitation (ChIP):
Research by Larsen et al. utilized ChIP with NBN antibodies to track recruitment patterns at site-specific DNA breaks:

• NBN accumulates directly at DNA break sites within 1 hour after damage induction

• Wild-type NBN peaks at 3 hours and then decreases by 5 hours

• Phospho-deficient mutants (S>A) show delayed recruitment, peaking at 5 hours

• Phospho-mimetic mutants (S>E) show prolonged accumulation up to 8 hours

This approach revealed that NBN phosphorylation regulates not only its own recruitment dynamics but also the recruitment of ATM to DNA damage sites, with important implications for DNA repair efficiency .

What controls should be included in experiments using NBN (Ab-343) Antibody?

Proper controls are essential for interpreting results with NBN (Ab-343) Antibody:

Positive Controls:

• Cell lines with known NBN expression (e.g., Jurkat, HeLa)

• Recombinant NBN protein (if available)

• Cells treated with DNA damaging agents (higher phosphorylation)

Negative Controls:

• Antibody pre-incubated with immunizing peptide (blocking peptide control)

• NBN-depleted samples (siRNA knockdown or CRISPR knockout)

• Secondary antibody only

Experimental Controls:

• For DNA damage studies: Paired untreated vs. treated samples

• For phosphorylation studies: Include λ-phosphatase-treated samples

• For cell cycle studies: Synchronized cell populations

Example from Validation Data:
Immunohistochemical analysis of human breast carcinoma tissue shows specific staining when using NBN (Ab-343) Antibody alone, while this staining is abolished when the antibody is pre-incubated with blocking peptide, confirming antibody specificity .

Including these controls ensures confidence in experimental results and facilitates troubleshooting if unexpected results occur.

What methodological approaches enable quantification of NBN phosphorylation kinetics?

To quantify NBN phosphorylation kinetics with precision:

Dual Antibody Approach:

• Use NBN (Ab-343) Antibody to measure total NBN levels

• Use phospho-specific antibodies (e.g., anti-p95/NBS1 phospho S343) for phosphorylated forms

• Calculate phosphorylation ratio (phospho-NBN/total NBN) to normalize for expression differences

Time-course Experimental Design:

Advanced Techniques for Phosphorylation Analysis:

• Phos-tag SDS-PAGE: Separates phosphorylated from non-phosphorylated forms based on mobility shift

• Mass spectrometry: Identifies multiple phosphorylation sites simultaneously and their relative abundances

• FRET-based sensors: Monitors phosphorylation in real-time in living cells

Data Analysis and Interpretation:

• Plot phosphorylation ratio over time to generate kinetic curves

• Calculate rate constants for phosphorylation and dephosphorylation

• Compare kinetics between wild-type and mutant systems

• Correlate with functional outcomes (e.g., DNA repair efficiency, checkpoint activation)

Research by Larsen et al. revealed that phosphorylation-mimicking mutations (S>E) resulted in both increased and prolonged accumulation of NBN at DNA breaks compared to wild-type, while phosphorylation-blocking mutations (S>A) delayed recruitment, demonstrating the regulatory role of phosphorylation in DNA damage response dynamics .

How can chromatin immunoprecipitation (ChIP) with NBN (Ab-343) Antibody be optimized for DNA damage studies?

Optimizing ChIP with NBN (Ab-343) Antibody for DNA damage studies requires careful experimental design:

Protocol Optimization for Site-Specific DNA Breaks:

• Induce site-specific DNA breaks using endonucleases like I-PpoI

• Fix cells with formaldehyde at multiple time points post-damage

• Sonicate chromatin to 200-500 bp fragments

• Immunoprecipitate with 2-5 μg NBN (Ab-343) Antibody

• Design primers for qPCR at various distances from break sites:

  • Directly at break site

  • 1-3 kb from break site

  • 5-10 kb from break site

  • 20+ kb from break site as background control

Key Parameters from Research Literature:

• NBN accumulates directly at DNA break sites, while ATM is detected in regions 3-10 kb flanking the site

• In wild-type cells, NBN is detected at the cleavage site within 1 hour after damage, peaks at 3 hours, and decreases by 5 hours

• At later time points, NBN accumulation shifts to sites 3-10 kb flanking the DSB

• Phosphorylation status significantly impacts both timing and extent of NBN accumulation

Controls and Validation:

• Input chromatin (5-10% of starting material)

• IgG immunoprecipitation as negative control

• Known positive loci (e.g., previously characterized DSB sites)

• Parallel ChIP with antibodies against other DNA damage response proteins (γH2AX, 53BP1)

Larsen et al. demonstrated that blocking NBN phosphorylation (S>A mutations) delayed recruitment to break sites, while phospho-mimetic mutations (S>E) caused prolonged accumulation, revealing the regulatory role of phosphorylation in the spatial and temporal dynamics of the DNA damage response .

How can NBN (Ab-343) Antibody be used in studies comparing DNA repair efficiency between cell lines?

NBN (Ab-343) Antibody can be integrated into comprehensive DNA repair efficiency studies:

Experimental Design for Comparative Repair Studies:

• Select cell lines of interest (e.g., normal vs. cancer, wildtype vs. mutant)

• Induce DNA damage (site-specific or global)

• Monitor repair kinetics over time (0, 1, 3, 5, 8, 24 hours)

• Use multiple readouts to comprehensively assess repair efficiency

Multi-parameter Analysis Workflow:

Insights from Research Literature:

• Larsen et al. found that cells lacking full-length NBN showed significantly delayed repair compared to control cells

• Blocking phosphorylation of NBN resulted in a more modest delay in repair

• Phosphorylation status affected both the timing of recruitment and the duration of NBN presence at break sites

• These findings establish NBN phosphorylation as a regulatory mechanism for DNA repair efficiency

By combining NBN (Ab-343) Antibody with these analytical approaches, researchers can generate comprehensive profiles of DNA repair capabilities across different cell types and genetic backgrounds, providing insights into repair deficiencies associated with cancer or other genomic instability syndromes.