Phospho-S6K1(T421/S424) Rabbit Recombinant mAb

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20ul RMB 547.33 现货
100ul RMB 1700.76 现货
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Cited by 1 Publication

使用信息

抗体应用 WB,ELISA
稀释比例
WB
1:1000
反应性 Human Rat
MW (kDa) 70kDa
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-S6K1(T421/S424) Rabbit Recombinant mAb可检测phospho-S6K1(T421/S424)的内源性水平。
背景 Ribosomal protein S6 kinase 1 (S6K1)是调节细胞生长的关键因子。S6K1的下游效应因子包括ribosomal S6 protein, eukaryotic initiation factor 4B, programmed cell death 4, eukaryotic elongation factor-2 kinase, mTOR, GSK3, insulin receptor substrate 1和 S6K1 Aly/REF-like target。S6K1有两种已知亚型:p85(S6K1)和p70(S6K1)。p85(S6K1)主要位于核内,而p70(S6K1)主要位于胞质。S6K1可被胰岛素和其他分裂素通过多位点磷酸化而激活。Ser411、Thr421、Ser424位于富含Ser-Pro区域。这些位点的磷酸化可激活p70(S6K1)。

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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