Phospho-p95/NBS1 (S343) Rabbit Recombinant mAb

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  • WB
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20ul 547.33 现货
100ul 1700.76 现货
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使用信息

抗体应用 WB,ELISA
稀释比例
WB
1:1000
反应性 Human
MW (kDa) 95kDa
抗体类型 Rabbit
浓度 1mg/ml
储存液配方 10 mM sodium HEPES (pH 7.5), 150 mM NaCl, 100 µg/ml BSA, 50% glycerol and less than 0.02% sodium azide.
储存条件
(自收到货起)
Store at –20°C.

Datasheet & SDS

生物描述

特异性 Phospho-p95/NBS1 (S343) Rabbit Recombinant mAb detects endogenous level of total Phospho-p95/NBS1 (S343).
背景 Nbs1, a member of the Mre11-RAD50-Nbs1 complex, is phosphorylated by ATM, the product of the ataxia-telangiectasia mutated gene and a member of the phosphatidylinositol 3-kinase–related family of serine-threonine kinases, in response to DNA double-strand breaks (DSBs) to regulate DNA damage checkpoints. Nbs1 plays an important role in activation of intra-S and G2/M cell-cycle checkpoints. Nbs1 can reciprocally modulate the phosphorylation of ATM. This leads to the activation of an intra–S-phase checkpoint by phosphorylation of SMC1 and/or FANCD2 or a G2/M checkpoint via the phosphorylations of Chk2 and the phosphatase Cdc25a. In addition to the regulation of checkpoints, Nbs1 may also play a direct role in the DSB repair by HRR and NHEJ. Nbs1 is phosphorylated on S343 by ATM in the S-phase checkpoint pathway and also has been demonstrated to modulate the activity of ATM.

实验方法

WB

Western Blotting

Sample preparation

1. Aspirate media from cultures and Wash the cells with 1X PBS.
2. Lyse cells by adding 1X SDS sample buffer and transfer the extract to a microcentrifuge tube. Keep onice.
3. Sonicate for 10–15 sec to complete cell lysis and shear DNA.
4. Heat a 20 µl sample to 95–100°C for 5 min, then cool on ice.
5. Centrifuge for 5 min (with Microcentrifuge).
6. Load appropriate volumes of samples onto SDS-PAGE gel (loading quantity of protein sample depends on the concentration of extracted proteins).
NOTE: At the same time, please load the pre-stained molecular weight markers to determine molecular weights and verify electrotransfer.
7. Electrotransfer to nitrocellulose/PVDF membrane.

Membrane Blocking and Antibody Incubations

a. Blocking

1. (Optional) After transfer, wash the transferred membrane with TBS for 5 min at room temperature.
2. Incubate the membrane in the blocking buffer for 1 hr at room temperature.
3. Wash three times for 5 min each with TBST.

b. Antibodies Incubation

1. Incubate membrane and primary antibody (at the appropriate dilution and diluent recommended) in a primary antibody dilution buffer with gentle agitation overnight at 4°C.
2. Wash three times for 5 min each with TBST.
3. Incubate membrane with an appropriate second antibodydissolved in the blocking buffer with gentle agitation for 1 hr at room temperature.
4. Wash three times for 5 min each with TBST.
5. Proceed with detection.

Detection of Proteins

1. After antibodies incubation, Wash membrane three times for 5 minutes in TBST.
2. PrepareECL Reagent (or other chromogenic agents/substrate according to your second antibody). Mix well.
3. Incubate substrate with membrane for 1 minute, remove excess solution (membrane remains wet), wrap in plastic and expose to X-ray film.

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