p95/NBS1 Antibody [P16D23] | Primary Antibodies

Application: Reactivity: Human

Lane 1: Hela, Lane 2: Jurkat

Usage Information

Dilution
WB1:1000 - 1:10000 IHC1:200

Application
WB, IHC

Reactivity
Human

Source
Rabbit Monoclonal Antibody

Storage Buffer
PBS, pH 7.2+50% Glycerol+0.05% BSA+0.01% NaN3

Storage (from the date of receipt)
-20°C (avoid freeze-thaw cycles), 2 years

Predicted MW Observed MW
84 kDa 90 kDa,95 kDa
^*Why do the predicted and actual molecular weights differ? The following reasons may explain differences between the predicted and actual protein molecular weight. Post-translational modifications(e.g., phosphorylation, glycosylation); Splice variants and isoforms; Relative charge; Multimerization.

Positive Control	Human testis tissue; A431 cells; HeLa cells; Jurkat cells
Negative Control

Experimental Methods

WB
Experimental Protocol: Sample preparation 1. Tissue: Lyse the tissue sample by adding an appropriate volume of ice-cold RIPA/Nuclear Lysis Buffer (containing Protease Inhibitor Cocktail)，and homogenize the tissue at a low temperature or lyse it by sonication on ice, then incubate on ice for 30 minutes. 2. Adherent cell: Aspirate the culture medium and wash the cells with ice-cold PBS twice. Lyse the cells by adding an appropriate volume of RIPA/Nuclear Lysis Buffer (containing Protease Inhibitor Cocktail) , sonicate to lyse the cells, and incubate on ice for 30 minutes. 3. Suspension cell: Transfer the culture medium to a pre-cooled centrifuge tube. Centrifuge and aspirate the supernatant. Wash the cells with ice-cold PBS twice. Lyse the cells by adding an appropriate volume of RIPA/Nuclear Lysis Buffer (containing Protease Inhibitor Cocktail) , sonicate to lyse the cells, and incubate on ice for 30 minutes. 4. Place the lysate into a pre-cooled microcentrifuge tube. Centrifuge at 4°C for 15 min. Collect the supernatant; 5. Remove a small volume of lysate to determine the protein concentration; 6. Combine the lysate with protein loading buffer. Boil 20 µL sample under 95-100°C for 5 min. Centrifuge for 5 min after cool down on ice. Electrophoretic separation 1. According to the concentration of extracted protein, load appropriate amount of protein sample and marker onto SDS-PAGE gels for electrophoresis. Recommended separating gel (lower gel) concentration: 10%. Reference Table for Selecting SDS-PAGE Separation Gel Concentrations 2. Power up 80V for 30 minutes. Then the power supply is adjusted (110 V~150 V), the Marker is observed, and the electrophoresis can be stopped when the indicator band of the predyed protein Marker where the protein is located is properly separated. (Note that the current should not be too large when electrophoresis, too large current (more than 150 mA) will cause the temperature to rise, affecting the result of running glue. If high currents cannot be avoided, an ice bath can be used to cool the bath.) Transfer membrane 1. Take out the converter, soak the clip and consumables in the pre-cooled converter; 2. Activate PVDF membrane with methanol for 1 min and rinse with transfer buffer; 3. Install it in the order of "black edge of clip - sponge - filter paper - filter paper - glue -PVDF membrane - filter paper - filter paper - sponge - white edge of clip"; 4. The protein was electrotransferred to PVDF membrane. ( 0.45 µm PVDF membrane is recommended ) Reference Table for Selecting PVDF Membrane Pore Size Specifications Recommended conditions for wet transfer: 200 mA, 120 min. ( Note that the transfer conditions can be adjusted according to the protein size. For high-molecular-weight proteins, a higher current and longer transfer time are recommended. However, ensure that the transfer tank remains at a low temperature to prevent gel melting.) Block 1. After electrotransfer, wash the film with TBST at room temperature for 5 minutes; 2. Incubate the film in the blocking solution for 1 hour at room temperature; 3. Wash the film with TBST for 3 times, 5 minutes each time. Antibody incubation 1. Use primary antibody dilution buffer to prepare the primary antibody working liquid (recommended dilution ratio for primary antibody 1:1000), gently shake and incubate with the film at 4°C overnight; 2. Wash the film with TBST 3 times, 5 minutes each time; 3. Add the secondary antibody to the blocking solution and incubate with the film gently at room temperature for 1 hour; 4. After incubation, wash the film with TBST 3 times for 5 minutes each time. Antibody staining 1. Add the prepared ECL luminescent substrate (or select other color developing substrate according to the second antibody) and mix evenly; 2. Incubate with the film for 1 minute, remove excess substrate (keep the film moist), wrap with plastic film, and expose in the imaging system.

WB

Experimental Protocol:

Sample preparation

1. Tissue: Lyse the tissue sample by adding an appropriate volume of ice-cold RIPA/Nuclear Lysis Buffer (containing Protease Inhibitor Cocktail)，and homogenize the tissue at a low temperature or lyse it by sonication on ice, then incubate on ice for 30 minutes.

2. Adherent cell: Aspirate the culture medium and wash the cells with ice-cold PBS twice. Lyse the cells by adding an appropriate volume of RIPA/Nuclear Lysis Buffer (containing Protease Inhibitor Cocktail) , sonicate to lyse the cells, and incubate on ice for 30 minutes.

3. Suspension cell: Transfer the culture medium to a pre-cooled centrifuge tube. Centrifuge and aspirate the supernatant. Wash the cells with ice-cold PBS twice. Lyse the cells by adding an appropriate volume of RIPA/Nuclear Lysis Buffer (containing Protease Inhibitor Cocktail) , sonicate to lyse the cells, and incubate on ice for 30 minutes.

4. Place the lysate into a pre-cooled microcentrifuge tube. Centrifuge at 4°C for 15 min. Collect the supernatant;

5. Remove a small volume of lysate to determine the protein concentration;

6. Combine the lysate with protein loading buffer. Boil 20 µL sample under 95-100°C for 5 min. Centrifuge for 5 min after cool down on ice.

Electrophoretic separation

1. According to the concentration of extracted protein, load appropriate amount of protein sample and marker onto SDS-PAGE gels for electrophoresis. Recommended separating gel (lower gel) concentration: 10%. Reference Table for Selecting SDS-PAGE Separation Gel Concentrations

2. Power up 80V for 30 minutes. Then the power supply is adjusted (110 V~150 V), the Marker is observed, and the electrophoresis can be stopped when the indicator band of the predyed protein Marker where the protein is located is properly separated. (Note that the current should not be too large when electrophoresis, too large current (more than 150 mA) will cause the temperature to rise, affecting the result of running glue. If high currents cannot be avoided, an ice bath can be used to cool the bath.)

Transfer membrane

1. Take out the converter, soak the clip and consumables in the pre-cooled converter;

2. Activate PVDF membrane with methanol for 1 min and rinse with transfer buffer;

3. Install it in the order of "black edge of clip - sponge - filter paper - filter paper - glue -PVDF membrane - filter paper - filter paper - sponge - white edge of clip";

4. The protein was electrotransferred to PVDF membrane. ( 0.45 µm PVDF membrane is recommended ) Reference Table for Selecting PVDF Membrane Pore Size Specifications

Recommended conditions for wet transfer: 200 mA, 120 min.

( Note that the transfer conditions can be adjusted according to the protein size. For high-molecular-weight proteins, a higher current and longer transfer time are recommended. However, ensure that the transfer tank remains at a low temperature to prevent gel melting.)

Block

1. After electrotransfer, wash the film with TBST at room temperature for 5 minutes;

2. Incubate the film in the blocking solution for 1 hour at room temperature;

3. Wash the film with TBST for 3 times, 5 minutes each time.

Antibody incubation

1. Use primary antibody dilution buffer to prepare the primary antibody working liquid (recommended dilution ratio for primary antibody 1:1000), gently shake and incubate with the film at 4°C overnight;

2. Wash the film with TBST 3 times, 5 minutes each time;

3. Add the secondary antibody to the blocking solution and incubate with the film gently at room temperature for 1 hour;

4. After incubation, wash the film with TBST 3 times for 5 minutes each time.

Antibody staining

1. Add the prepared ECL luminescent substrate (or select other color developing substrate according to the second antibody) and mix evenly;

2. Incubate with the film for 1 minute, remove excess substrate (keep the film moist), wrap with plastic film, and expose in the imaging system.

IHC
Experimental Protocol： Deparaffinization/Rehydration 1. Deparaffinize/hydrate sections: 2. Incubate sections in three washes of xylene for 5 min each. 3. Incubate sections in two washes of 100% ethanol for 10 min each. 4. Incubate sections in two washes of 95% ethanol for 10 min each. 5. Wash sections two times in dH2O for 5 min each. 6.Antigen retrieval: For Citrate: Heat slides in a microwave submersed in 1X citrate unmasking solution until boiling is initiated; continue with 10 min at a sub-boiling temperature (95°-98°C). Cool slides on bench top for 30 min. Staining 1. Wash sections in dH2O three times for 5 min each. 2. Incubate sections in 3% hydrogen peroxide for 10 min. 3. Wash sections in dH2O two times for 5 min each. 4. Wash sections in wash buffer for 5 min. 5. Block each section with 100–400 µl of blocking solution for 1 hr at room temperature. 6. Remove blocking solution and add 100–400 µl primary antibody diluent in to each section. Incubate overnight at 4°C. 7. Remove antibody solution and wash sections with wash buffer three times for 5 min each. 8. Cover section with 1–3 drops HRPas needed. Incubate in a humidified chamber for 30 min at room temperature. 9. Wash sections three times with wash buffer for 5 min each. 10. Add DAB Chromogen Concentrate to DAB Diluent and mix well before use. 11. Apply 100–400 µl DAB to each section and monitor closely. 1–10 min generally provides an acceptable staining intensity. 12. Immerse slides in dH2O. 13. If desired, counterstain sections with hematoxylin. 14. Wash sections in dH2O two times for 5 min each. 15. Dehydrate sections: Incubate sections in 95% ethanol two times for 10 sec each; Repeat in 100% ethanol, incubating sections two times for 10 sec each; Repeat in xylene, incubating sections two times for 10 sec each. 16. Mount sections with coverslips and mounting medium.

IHC

Experimental Protocol：

Deparaffinization/Rehydration

1. Deparaffinize/hydrate sections:

2. Incubate sections in three washes of xylene for 5 min each.

3. Incubate sections in two washes of 100% ethanol for 10 min each.

4. Incubate sections in two washes of 95% ethanol for 10 min each.

5. Wash sections two times in dH2O for 5 min each.

6.Antigen retrieval: For Citrate: Heat slides in a microwave submersed in 1X citrate unmasking solution until boiling is initiated; continue with 10 min at a sub-boiling temperature (95°-98°C). Cool slides on bench top for 30 min.

Staining

1. Wash sections in dH2O three times for 5 min each.

2. Incubate sections in 3% hydrogen peroxide for 10 min.

3. Wash sections in dH2O two times for 5 min each.

4. Wash sections in wash buffer for 5 min.

5. Block each section with 100–400 µl of blocking solution for 1 hr at room temperature.

6. Remove blocking solution and add 100–400 µl primary antibody diluent in to each section. Incubate overnight at 4°C.

7. Remove antibody solution and wash sections with wash buffer three times for 5 min each.

8. Cover section with 1–3 drops HRPas needed. Incubate in a humidified chamber for 30 min at room temperature.

9. Wash sections three times with wash buffer for 5 min each.

10. Add DAB Chromogen Concentrate to DAB Diluent and mix well before use.

11. Apply 100–400 µl DAB to each section and monitor closely. 1–10 min generally provides an acceptable staining intensity.

12. Immerse slides in dH2O.

13. If desired, counterstain sections with hematoxylin.

14. Wash sections in dH2O two times for 5 min each.

15. Dehydrate sections: Incubate sections in 95% ethanol two times for 10 sec each; Repeat in 100% ethanol, incubating sections two times for 10 sec each; Repeat in xylene, incubating sections two times for 10 sec each.

16. Mount sections with coverslips and mounting medium.

Datasheet & SDS

Biological Description

Specificity
p95/NBS1 Antibody (Rabbit mAb) [P16D23] detects endogenous levels of total p95/NBS1 protein.

Subcellular Location
Chromosome, Nucleus, Telomere

Uniprot ID
O60934

Clone
P16D23

Synonym(s)
NBS, NBS1, P95, NBN, Nibrin, Cell cycle regulatory protein p95, Nijmegen breakage syndrome protein 1, hNbs1

Background
p95/NBS1 (nibrin, NBN) is a phosphoprotein component of the MRE11–RAD50–NBS1 (MRN) complex that couples DNA double‑strand break (DSB) recognition to ATM activation, checkpoint signaling, and high‑fidelity repair, thereby acting as a key organizer of the DNA damage response and a guardian of chromosomal integrity. The N‑terminal region contains an FHA domain followed by tandem BRCT repeats that bind phosphorylated epitopes on mediator proteins such as MDC1 and γH2AX, and this phospho‑recognition module is essential for recruiting and retaining the MRN complex at DSB sites and for proper G2/M checkpoint control, while the C‑terminal part mediates interaction with MRE11, RAD50, and additional partners, including ATM and the p110α subunit of PI3K. Upon DSB formation, NBS1 directs MRN accumulation to chromatin via γH2AX binding and positions the complex to stimulate ATM activation; ATM phosphorylates NBS1 at defined serines in an S‑phase checkpoint module, and NBS1 in turn amplifies ATM signaling by concentrating ATM and its substrates at damage foci, resulting in coordinated phosphorylation of downstream effectors such as CHK2, p53, and SMC1 that enforce S‑phase slowing, G2/M arrest, and transcriptional responses while repair proceeds. The MRN–NBS1 assembly also promotes DNA end tethering, processing, and channeling into homologous recombination and, to a lesser extent, modulates non‑homologous end joining, and NBS1 has additional roles in suppressing aberrant inter‑chromosomal recombination and maintaining telomere stability through effects on TRF2 and ATM‑dependent telomere damage signaling, so that NBS1 deficiency leads to impaired checkpoint activation, defective DSB repair, accelerated telomere erosion, telomere fusions, and aneuploidy. NBS1 can act as an adaptor for growth signaling: a conserved C‑terminal motif directly binds the N‑terminal region of p110α and stimulates class I PI3K activity, and NBS1 overexpression activates PI3K–Akt signaling and induces cellular transformation. Deregulated NBS1 can couple DNA damage pathways to oncogenic survival and proliferation circuits. Germline loss‑of‑function mutations in NBS1 cause Nijmegen breakage syndrome, a recessive chromosomal instability disorder characterized by microcephaly, growth retardation, immunodeficiency, radiosensitivity, and a high incidence of lymphoid malignancies, and heterozygous carriers of recurrent mutations such as 657del5 show increased breast cancer risk, reflecting the dose‑dependent requirement of NBS1 for genome maintenance.

Background

p95/NBS1 (nibrin, NBN) is a phosphoprotein component of the MRE11–RAD50–NBS1 (MRN) complex that couples DNA double‑strand break (DSB) recognition to ATM activation, checkpoint signaling, and high‑fidelity repair, thereby acting as a key organizer of the DNA damage response and a guardian of chromosomal integrity. The N‑terminal region contains an FHA domain followed by tandem BRCT repeats that bind phosphorylated epitopes on mediator proteins such as MDC1 and γH2AX, and this phospho‑recognition module is essential for recruiting and retaining the MRN complex at DSB sites and for proper G2/M checkpoint control, while the C‑terminal part mediates interaction with MRE11, RAD50, and additional partners, including ATM and the p110α subunit of PI3K. Upon DSB formation, NBS1 directs MRN accumulation to chromatin via γH2AX binding and positions the complex to stimulate ATM activation; ATM phosphorylates NBS1 at defined serines in an S‑phase checkpoint module, and NBS1 in turn amplifies ATM signaling by concentrating ATM and its substrates at damage foci, resulting in coordinated phosphorylation of downstream effectors such as CHK2, p53, and SMC1 that enforce S‑phase slowing, G2/M arrest, and transcriptional responses while repair proceeds. The MRN–NBS1 assembly also promotes DNA end tethering, processing, and channeling into homologous recombination and, to a lesser extent, modulates non‑homologous end joining, and NBS1 has additional roles in suppressing aberrant inter‑chromosomal recombination and maintaining telomere stability through effects on TRF2 and ATM‑dependent telomere damage signaling, so that NBS1 deficiency leads to impaired checkpoint activation, defective DSB repair, accelerated telomere erosion, telomere fusions, and aneuploidy. NBS1 can act as an adaptor for growth signaling: a conserved C‑terminal motif directly binds the N‑terminal region of p110α and stimulates class I PI3K activity, and NBS1 overexpression activates PI3K–Akt signaling and induces cellular transformation. Deregulated NBS1 can couple DNA damage pathways to oncogenic survival and proliferation circuits. Germline loss‑of‑function mutations in NBS1 cause Nijmegen breakage syndrome, a recessive chromosomal instability disorder characterized by microcephaly, growth retardation, immunodeficiency, radiosensitivity, and a high incidence of lymphoid malignancies, and heterozygous carriers of recurrent mutations such as 657del5 show increased breast cancer risk, reflecting the dose‑dependent requirement of NBS1 for genome maintenance.

References
https://pubmed.ncbi.nlm.nih.gov/15279770/ https://pubmed.ncbi.nlm.nih.gov/18270679/

Protein Size (kDa)	PVDF Transfer Membrane Pore Size (μm)
< 10	0.22
10-20	0.22
20-30	0.45
30-45	0.45
45-70	0.45
80-100	0.45
100-140	0.45
> 140	0.45

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Protein Size (kDa)	Separating Gel Percentage
4-40	20%
12-45	15%
10-70	12.5%
15-100	10%
25-100	8%
60-210	5%

p95/NBS1 Antibody (Rabbit mAb) [P16D23]

Usage Information

Experimental Methods

Datasheet & SDS

Biological Description

Tech Support