ERK1/2 Rabbit Recombinant mAb

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20ul RMB 447.16 现货
100ul RMB 1500.76 现货
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Cited by 3 Publications

使用信息

抗体应用 WB,ELISA
稀释比例
WB
1:2000
反应性 Human Mouse Rat
MW (kDa) 42kDa
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

ERK1/2 Rabbit Recombinant mAb detects endogenous levels of total ERK1/2.

背景

ERK1 and ERK2 are related protein-serine/threonine kinases that participate in the Ras-Raf-MEK-ERK signal transduction cascade. This cascade participates in the regulation of a large variety of processes including cell adhesion, cell cycle progression, cell migration, cell survival, differentiation, metabolism, proliferation, and transcription. MEK1/2 catalyze the phosphorylation of human ERK1/2 at Tyr204/187 and then Thr202/185. The phosphorylation of both tyrosine and threonine is required for enzyme activation. The ERK1/2 catalyzed phosphorylation of nuclear transcription factors including those of Ets, Elk, and c-Fos represents an important function and requires the translocation of ERK1/2 into the nucleus by active and passive processes involving the nuclear pore. These transcription factors participate in the immediate early gene response.

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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