Human NBN (p95/NBS1) knockout A549 cell line (ab276103)
製品の概要
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製品名
Human NBN (p95/NBS1) knockout A549 cell line
p95/NBS1 ライゼート 製品一覧 -
Parental Cell Line
A549 -
Organism
Human -
Mutation description
Knockout achieved by using CRISPR/Cas9, Homozygous: 132 bp deletion in exon 2 and Intron 2-3. -
Passage number
<20 -
Knockout validation
Western Blot (WB) -
Biosafety level
1 -
特記事項
Recommended control: Human wild-type A549 cell line (ab275463). Please note a wild-type cell line is not automatically included with a knockout cell line order, if required please add recommended wild-type cell line at no additional cost using the code WILDTYPE-TMTK1.
Cryopreservation cell medium: Cell Freezing Medium-DMSO Serum free media, contains 8.7% DMSO in MEM supplemented with methyl cellulose.
Culture medium: F-12K + 10% FBS
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. Based on cell count, seed cells in an appropriate cell culture flask at a density of 2x103-1x104 cells/cm2. Seeding density is given as a guide only and should be scaled to align with individual lab schedules.
4. Incubate the culture at 37°C incubator with 5% CO2. 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 6x104 cells/cm2 is recommended.
- A partial media change 24 hours prior to subculture may be helpful to encourage growth, if required.
- Cells should be passaged when they have achieved 80-90% confluence.
- Do not exceed 7x104 cells/cm2.
This product is subject to limited use licenses from The Broad Institute and ERS Genomics Limited, and is developed with patented technology. For full details of the limited use licenses and relevant patents please refer to our limited use license and patent pages.
We will provide viable cells that proliferate on revival.
製品の特性
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Number of cells
1 x 106 cells/vial, 1 mL -
Viability
~50% -
Adherent /Suspension
Adherent -
Tissue
Lung -
Cell type
epithelial -
Disease
Carcinoma -
Gender
Male -
Mycoplasma free
Yes -
保存方法
Shipped on Dry Ice. Store in liquid nitrogen. -
バッファー
Constituents: 8.7% Dimethylsulfoxide, 2% Cellulose, methyl ether -
研究分野
ターゲット情報
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機能
Component of the MRE11-RAD50-NBN (MRN complex) which plays a critical role in the cellular response to DNA damage and the maintenance of chromosome integrity. The complex is involved in double-strand break (DSB) repair, DNA recombination, maintenance of telomere integrity, cell cycle checkpoint control and meiosis. The complex possesses single-strand endonuclease activity and double-strand-specific 3'-5' exonuclease activity, which are provided by MRE11A. RAD50 may be required to bind DNA ends and hold them in close proximity. NBN modulate the DNA damage signal sensing by recruiting PI3/PI4-kinase family members ATM, ATR, and probably DNA-PKcs to the DNA damage sites and activating their functions. It can also recruit MRE11 and RAD50 to the proximity of DSBs by an interaction with the histone H2AX. NBN also functions in telomere length maintenance by generating the 3' overhang which serves as a primer for telomerase dependent telomere elongation. NBN is a major player in the control of intra-S-phase checkpoint and there is some evidence that NBN is involved in G1 and G2 checkpoints. The roles of NBS1/MRN encompass DNA damage sensor, signal transducer, and effector, which enable cells to maintain DNA integrity and genomic stability. Forms a complex with RBBP8 to link DNA double-strand break sensing to resection. Enhances AKT1 phosphorylation possibly by association with the mTORC2 complex. -
組織特異性
Ubiquitous. Expressed at high levels in testis. -
関連疾患
Nijmegen breakage syndrome
Breast cancer
Aplastic anemia
Defects in NBN might play a role in the pathogenesis of childhood acute lymphoblastic leukemia (ALL). -
配列類似性
Contains 1 BRCT domain.
Contains 1 FHA domain. -
ドメイン
The FHA and BRCT domains are likely to have a crucial role for both binding to histone H2AFX and for relocalization of MRE11/RAD50 complex to the vicinity of DNA damage.
The C-terminal domain contains a MRE11-binding site, and this interaction is required for the nuclear localization of the MRN complex.
The EEXXXDDL motif at the C-terminus is required for the interaction with ATM and its recruitment to sites of DNA damage and promote the phosphorylation of ATM substrates, leading to the events of DNA damage response. -
翻訳後修飾
Phosphorylated by ATM in response of ionizing radiation, and such phosphorylation is responsible intra-S phase checkpoint control and telomere maintenance. -
細胞内局在
Nucleus. Nucleus, PML body. Chromosome, telomere. Localizes to discrete nuclear foci after treatment with genotoxic agents. - Information by UniProt
画像
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Western blot - Human NBN (p95/NBS1) knockout A549 cell line (ab276103)All lanes : Anti-p95/NBS1 antibody [7E4C2] (ab181780) at 1/500 dilution
Lane 1 : Wild-type A549 cell lysate
Lane 2 : NBN knockout A549 cell lysate
Lane 3 : Wild-type A431 cell lysate
Lane 4 : NBN knockout A431 cell lysate
Lysates/proteins at 20 µg per lane.
Performed under reducing conditions.
Observed band size: 95 kDa why is the actual band size different from the predicted?Anti-NBN antibody [7E4C2] (ab181780) 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 NBN. A band was observed at 95 kDa in wild-type A549 cell lysates with no signal observed at this size in NBN knockout cell line. To generate this image, wild-type and NBN knockout A549 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 °C. 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 800CW and Goat anti-Rabbit IgG H&L 680RD at 1/20000 dilution.
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Sanger Sequencing - Human NBN (p95/NBS1) knockout A549 cell line (ab276103)132 bp deletion in exon 2 and Intron 2-3.
プロトコール
データシートおよび資料
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SDS download
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Datasheet download
参考文献 (0)
ab276103 は論文での使用が確認できていません。