磷酸化Akt(Ser473)检测试剂盒(基于细胞荧光检测)
Phospho-Akt (Ser473) Detection Kit A (Cell-based Fluorimetric Assay)

Akt plays an essential role in cell survival, growth, migration, proliferation, polarity, and metabolism (lipid and glucose). Akt is activated through receptor tyrosine kinase pathways. Thr308 and Ser473 phosphorylation are necessary and sufficient for the full activation of Akt. Quantification of intracellular proteins and phosphorylation events is extremely important for biomedical research. The plate-based immunoassay has become a popular alternative method for rapid protein detection.

The ABSBio™ Phospho-Akt(Ser473) Fluorimetric Detection Kit provides a Cell-based ELISA format allows two target cellular proteins, or events, to be analyzed simultaneously in the same well. Cells are grown in 96-well plates and treated with the appropriate conditions, such as inhibitors or ligand stimulation. The cells are then fixed and permeabilized in the wells. This is followed by incubation with two primary antibodies derived from different species: a phospho-specific antibody (pAkt-Ser473) and a normalization antibody (Akt) that recognizes the total protein regardless of its phosphorylation status. Species-specific secondary antibodies labeled with horseradish peroxidase (HRP) and alkaline phosphatase (AP), and spectrally distinct fluorogenic substrates for each enzyme, are used for detection. The fluorescence of the phosphorylated protein is normalized to that of the total protein in each well to correct for well-to-well variations. The kit provides reagents sufficient for 100 assays (96-well plate). Cell-Based ELISAs are amenable to high-throughput applications and may prove a valuable addition to kinase inhibitor screening strategies.

试剂盒组分:

Storage and Handling: Store kit at -20°C, protected from light. Shelf life: 6 months after receipt. Warm Reagents to room temperature before use.

应用:Measurement of Akt phosphorylation level in whole cells, Cell based screening kinase inhibitors and ligand stimulation.

反应物种:Human, Mouse, Rat

样品类型:Culture cell lines-whole cell assay

检测方法:Fluorimetric cell based ELISA assay, (Ex/Em=530/590 nm; 360/450 nm)

时间:3-4 hrs

存储:-20°C

保质期:6 months after receipt.

运输:Icepacks

所需其他材料:37% formaldehyde (Molecular Biology Grade; Sigma, Catalog # F8775), refer to MSDS prior to use 3% H2O2 (Sigma, Catalog # 323381). refer to MSDS prior to use Tissue culture Black clear-bottom Corning 96 well plate (Sigma, cat# CLS3603). Multi-channel pipette for washing Cell culture incubator Orbital shaker Fluorescence plate reader with two channels: excitation 530 nm/emission 590 nm and excitation 360 nm/emission 450 nm