14-3-3 epsilon Rabbit Recombinant mAb

目录号：A5681

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20ul	447.74	现货
100ul	1500.74	现货
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使用信息

抗体应用

WB ,ELISA

稀释比例

1:1000

反应性

Human Mouse Rat

MW (kDa)

29kDa

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	14-3-3 epsilon Rabbit Recombinant mAb detects endogenous level of total 14-3-3 epsilon.
背景	The 14-3-3 proteins are a family of conserved regulatory molecules expressed in all eukaryotic cells. They could bind a multitude of functionally diverse signaling proteins, including kinases, phosphatases, and transmembrane receptors and plays important roles in a wide range of vital regulatory processes, such as mitogenic signal transduction, apoptotic cell death, and cell cycle control. 14-3-3 is present in almost all tissues, including testes, liver, and heart. Within a eukaryotic cell, 14-3-3 is largely found in the cytoplasmic compartment. However, 14-3-3 proteins can also be detected at the plasma membrane and in intracellular organelles such as the nucleus and the Golgi apparatus. As a member of the 14-3-3 protein family, 14-3-3ε may integrate a number of signaling pathways by interacting with distinct proteins; therefore, 14-3-3ε serves functions in the regulation of the cell cycle and apoptosis. It binds with heat shock factor 1 (HSF1) through the extracellular signal regulated kinase/glycogen synthase kinase 3/14-3-3ε pathway, thus suppressing HSF1 activity during cellular proliferation. Inhibiting the interaction of 14-3-3ε with chloride intracellular channel protein 4 enhanced autophagy and contributed to mitochondrial and ER stress-induced apoptosis under starvation conditions. In addition, 14-3-3ε has an association with the occurrence and development of a number of types of tumor; for example, the expression of 14-3-3ε has been identified to be downregulated in gastric cancer In addition, 14-3-3ε has an association with the occurrence and development of a number of types of tumor; for example, the expression of 14-3-3ε has been identified to be downregulated in gastric cancer and pediatric astrocytoma, whereas it is upregulated in hepatocellular carcinoma, renal cancer and breast cancer. 14-3-3ε is essential for normal brain development and neuronal migration in the mouse. This function of 14-3-3ε seems to be accomplished at least in part by binding to CDK5/p35 phosphorylated NUDEL to maintain NUDEL phosphorylation.

特异性

14-3-3 epsilon Rabbit Recombinant mAb detects endogenous level of total 14-3-3 epsilon.

背景

The 14-3-3 proteins are a family of conserved regulatory molecules expressed in all eukaryotic cells. They could bind a multitude of functionally diverse signaling proteins, including kinases, phosphatases, and transmembrane receptors and plays important roles in a wide range of vital regulatory processes, such as mitogenic signal transduction, apoptotic cell death, and cell cycle control. 14-3-3 is present in almost all tissues, including testes, liver, and heart. Within a eukaryotic cell, 14-3-3 is largely found in the cytoplasmic compartment. However, 14-3-3 proteins can also be detected at the plasma membrane and in intracellular organelles such as the nucleus and the Golgi apparatus. As a member of the 14-3-3 protein family, 14-3-3ε may integrate a number of signaling pathways by interacting with distinct proteins; therefore, 14-3-3ε serves functions in the regulation of the cell cycle and apoptosis. It binds with heat shock factor 1 (HSF1) through the extracellular signal regulated kinase/glycogen synthase kinase 3/14-3-3ε pathway, thus suppressing HSF1 activity during cellular proliferation. Inhibiting the interaction of 14-3-3ε with chloride intracellular channel protein 4 enhanced autophagy and contributed to mitochondrial and ER stress-induced apoptosis under starvation conditions. In addition, 14-3-3ε has an association with the occurrence and development of a number of types of tumor; for example, the expression of 14-3-3ε has been identified to be downregulated in gastric cancer In addition, 14-3-3ε has an association with the occurrence and development of a number of types of tumor; for example, the expression of 14-3-3ε has been identified to be downregulated in gastric cancer and pediatric astrocytoma, whereas it is upregulated in hepatocellular carcinoma, renal cancer and breast cancer. 14-3-3ε is essential for normal brain development and neuronal migration in the mouse. This function of 14-3-3ε seems to be accomplished at least in part by binding to CDK5/p35 phosphorylated NUDEL to maintain NUDEL phosphorylation.

实验方法

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.

参考文献	[1] Fu H, et al. Annu Rev Pharmacol Toxicol. 2000, 40:617-47. [2] Lu K, et al. Oncol Lett. 2018, 5(1):338-346. [3] Toyo-oka K, et al. Nat Genet. 2003, 34(3):274-85.

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