Human NBN (p95/NBS1) knockout A-431 cell line
- Advanced Validation
- What is this?
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(0 Publication)
- WB
Lab
Western blot - Human NBN (p95/NBS1) knockout A-431 cell line (AB269506)
False colour image of Western blot : Anti-p95/NBS1 antibody [7E4A2] staining at 1/500 dilution shown in green; Rabbit Anti-GAPDH antibody [EPR16891] (ab181602) loading control staining at 1/20000 dilution shown in red. In Western blot ab181729 was shown to bind specifically to p95/NBS1. A band was observed at 95 kDa in wild-type A431 cell lysates with no signal observed at this size in NBN knockout cell line ab269506 (knockout cell lysate ab269668). To generate this image wild-type and NBN knockout A431 cell lysates were analysed. First samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3 % milk in TBS-0.1 % Tween® 20 (TBS-T) before incubation with primary antibodies overnight at 4°. Blots were washed four times in TBS-T incubated with secondary antibodies for 1 h at room temperature washed again four times then imaged.Secondary antibodies used were Goat anti-Mouse IgG H&L (IRDye® 800CW) preabsorbed (ab216772) and Goat anti-Rabbit IgG H&L (IRDye® 680RD) preabsorbed (ab216777) at 1/20000 dilution.
All lanes:
Western blot - Anti-p95/NBS1 antibody [7E4A2] (<a href='/en-us/products/primary-antibodies/p95-nbs1-antibody-7e4a2-ab181729'>ab181729</a>) at 1/500 dilution
Lane 1:
Wild-type A431 cell lysate at 20 µg
Lane 2:
NBN knockout A431 cell lysate at 20 µg
Lane 2:
Western blot - Human NBN (p95/NBS1) knockout A-431 cell line (ab269506)
Predicted band size: 84 kDa
Observed band size: 95 kDa
false
- WB
Lab
Western blot - Human NBN (p95/NBS1) knockout A-431 cell line (AB269506)
False colour image of Western blot : Anti-p95/NBS1 antibody [7E4C2] staining at 1/500 dilution shown in green; Rabbit Anti-GAPDH antibody [EPR16891] (ab181602) loading control staining at 1/20000 dilution shown in red. In Western blot ab181780 was shown to bind specifically to p95/NBS1. A band was observed at 95 kDa in wild-type A431 cell lysates with no signal observed at this size in NBN knockout cell line ab269506 (knockout cell lysate ab269668). To generate this image wild-type and NBN knockout A431 cell lysates were analysed. First samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3 % milk in TBS-0.1 % Tween® 20 (TBS-T) before incubation with primary antibodies overnight at 4°. Blots were washed four times in TBS-T incubated with secondary antibodies for 1 h at room temperature washed again four times then imaged.Secondary antibodies used were Goat anti-Mouse IgG H&L (IRDye® 800CW) preabsorbed (ab216772) and Goat anti-Rabbit IgG H&L (IRDye® 680RD) preabsorbed (ab216777) at 1/20000 dilution.
All lanes:
Western blot - Anti-p95/NBS1 antibody [7E4C2] (<a href='/en-us/products/primary-antibodies/p95-nbs1-antibody-7e4c2-ab181780'>ab181780</a>) at 1/500 dilution
Lane 1:
Wild-type A431 cell lysate at 20 µg
Lane 2:
NBN knockout A431 cell lysate at 20 µg
Lane 2:
Western blot - Human NBN (p95/NBS1) knockout A-431 cell line (ab269506)
Predicted band size: 84 kDa
Observed band size: 95 kDa
false
- WB
Lab
Western blot - Human NBN (p95/NBS1) knockout A-431 cell line (AB269506)
Lanes 1 - 2 : Merged signal (red and green). Green - ab32074 observed at 90 kDa. Red - loading control ab8245 (Mouse anti-GAPDH antibody [6C5]) observed at 37 kDa.
ab32074 was shown to react with p95/NBS1 in wild-type A431 cells in Western blot with loss of signal observed in NBN knockout cell line ab269506 (NBN knockout cell lysate ab269668). Wild-type A431 and NBN knockout cell lysates were subjected to SDS-PAGE. Membranes were blocked in 5 % milk in TBS-T (0.1 % Tween®) before incubation with ab32074 and ab8245 (Mouse anti-GAPDH antibody [6C5]) overnight at 4 ° at a 1 in 1000 dilution and a 1 in 20000 dilution respectively. Blots were incubated with Goat anti-Rabbit IgG H&L (IRDye® 800CW) preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preabsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 h at room temperature before imaging.
Lanes 1 - 2:
Western blot - Anti-p95/NBS1 antibody [Y112] (<a href='/en-us/products/primary-antibodies/p95-nbs1-antibody-y112-ab32074'>ab32074</a>) at 1/1000 dilution
Lanes 1 - 3:
Western blot - Anti-p95/NBS1 antibody (<a href='/en-us/products/primary-antibodies/p95-nbs1-antibody-ab23996'>ab23996</a>) at 1/1000 dilution
Lane 1:
Wild-type A431 cell lysate at 20 µg
Lanes 2 - 3:
NBN knockout A431 cell lysate at 20 µg
Lane 2:
Western blot - Human NBN (p95/NBS1) knockout A-431 cell line (ab269506)
Predicted band size: 84 kDa
Observed band size: 90 kDa,95 kDa
false
- NGS
Supplier Data
Next Generation Sequencing - Human NBN (p95/NBS1) knockout A-431 cell line (AB269506)
Knockout achieved by CRISPR/Cas9; X = 1 bp insertion, 2 bp deletion, 11 bp deletion; Frameshift : 99%
- NGS
Supplier Data
Next Generation Sequencing - Human NBN (p95/NBS1) knockout A-431 cell line (AB269506)
1 bp insertion after Tyr15 (allele 1), 11 bp deletion after Pro14 (allele 2), and 2 bp deletion after Tyr15 (allele 3) of the WT protein
Complementary Products
Primary Antibodies
AB32074
RabMAb|Recombinant|KO Validated|20ul selling size
![Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-p95/NBS1 antibody [Y112] (AB32074)](https://content.abcam.com/products/images/p95-nbs1-antibody-y112-ab32074--immunohistochemistry-formalin-pfa-fixed-paraffin-embedded-sections-img128052.jpg)
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Reactivity data
Product details
We will provide viable cells that proliferate on revival.
This product is subject to limited use licenses from The Broad Institute, ERS Genomics Limited and Sigma-Aldrich Co. LLC, and is developed with patented technology. For full details of the licenses and patents please refer to our limited use license and patent pages.
What's included?
Properties and storage information
Gene name
Gene editing type
Gene editing method
Knockout validation
Shipped at conditions
Appropriate short-term storage conditions
Appropriate long-term storage conditions
Handling procedures
Initial handling guidelines
Upon arrival, the vial should be stored in liquid nitrogen vapor phase and not at -80°C. Storage at -80°C may result in loss of viability.
1. Thaw the vial in 37°C water bath for approximately 1-2 minutes.
2. Transfer the cell suspension (0.8 mL) to a 15 mL/50 mL conical sterile polypropylene centrifuge tube containing 8.4 mL pre-warmed culture medium, wash vial with an additional 0.8 mL culture medium (total volume 10 mL) to collect remaining cells, and centrifuge at 201 x g (rcf) for 5 minutes at room temperature. 10 mL represents minimum recommended dilution. 20 mL represents maximum recommended dilution.
3. Resuspend the cell pellet in 5 mL pre-warmed culture medium and count using a haemocytometer or alternative cell counting method seed all remaining cells into a T25.
4. Incubate the culture at 37°C incubator with 5% CO2. Check the culture one day after revival and continue to check until 80% confluent. Media change can be given if needed.
5. Once confluent passage into an appropriate flask at a density of 2x104 cells/cm2. Seeding density is given as a guide only and should be scaled to align with individual lab schedules. Cultures should be monitored daily.
Subculture guidelines
- All seeding densities should be based on cell counts gained by established methods.
- A guide seeding density of 2x104 cells/cm2 is recommended.
- Cells should be passaged when they have achieved 80-90% confluence.
Culture medium
DMEM (High Glucose) + 10% FBS
Cryopreservation medium
Cell Freezing Medium-DMSO Serum free media, contains 8.7% DMSO in MEM supplemented with methyl cellulose.
Supplementary information
This supplementary information is collated from multiple sources and compiled automatically.
Biological function summary
The NBS1 protein functions as an essential component of the MRN complex which also includes MRE11 and RAD50 proteins. This complex is fundamental in accurately detecting DNA double-strand breaks and initiating repair processes. Through its actions NBS1 facilitates numerous cellular processes that preserve genomic integrity such as homologous recombination and non-homologous end joining. Its influence in controlling the cell cycle further emphasizes its role in maintaining cellular health.
Pathways
The NBS1 protein engages significantly in the DNA damage response and repair pathways notably impacting the ATM signaling pathway. By interacting with proteins such as ATM kinase NBS1 facilitates the phosphorylation and activation of several downstream effectors necessary for DNA repair and cell cycle checkpoints. The MRN complex also links with the RAD50 protein highlighting its involvement in these pathways.
Quality control
STR analysis
CSF1PO, D13S317, D7S820, D5S818, TH01, D16S539, TPOX
Cell culture
Biosafety level
EU: 1 US: 1
Adherent/suspension
Adherent
Gender
Female
Target data
Product promise
Please note: All products are 'FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC OR THERAPEUTIC PROCEDURES'.
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