SEPARASE (Phospho-Ser801) ELISA Kit

Sample

cultured cells

Species Reactivity

Human; Mouse

Detection Method

iELISA

Intended Use

The SEPARASE (Phospho-Ser801) Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can monitor SEPARASE protein phosphorylation and expression profile in cells. The kit can be used for measuring the relative amounts of phosphorylated SEPARASE in cultured cells as well as screening for the effects that various treatments, inhibitors (ie. siRNA or chemicals), or activators have on SEPARASE phosphorylation.

Storage

Upon receipt, the kit should be stored at 4°C. The un-opened kit will be stable for up to 6 months from the date of shipment if stored at 4°C. Diluted Anti-Phospho-SEPARASE (Ser801) Antibody, SEPARASE Antibody and diluted Anti- GAPDH Antibody can each be stored at 4°C for up to two weeks. HRP- Conjugated Anti-Rabbit IgG Antibody and HRP-Conjugated Anti-Mouse IgG Antibody will be stable at 4°C for up to six months. The SDS Solution and Quenching Buffer should be stored at room temperature or warmed up to room temperature if stored at 4°C.

General Description

The Colorimetric Cell-Based ELISA Kit allows for the detection of various target proteins and the effects that certain stimulation conditions have on target protein expression in different cell lines. Qualitative determination of target protein concentration is achieved by an indirect ELISA format. In essence, the target protein is captured by target-specific primary (1°) antibodies while the HRP-conjugated secondary (2°) antibodies bind the Fc region of the 1° antibody. Through this binding, the HRP enzyme conjugated to the 2° antibody can catalyze a colorimetric reaction upon substrate addition.
Due to the qualitative nature of the Cell-Based ELISA, multiple normalization methods are described: 1) a monoclonal antibody specific for human GAPDH is included to serve as an internal positive control in normalizing the target absorbance values. 2) Following the colorimetric measurement of HRP activity via substrate addition, the Crystal Violet wholecell staining method is used to determine cell density. After staining, the results can be analyzed by normalizing the absorbance values to cell amounts, by which the plating difference can be adjusted. 3) If a phosphorylated target is being detected, an antibody against the nonphosphorylated counterpart will be provided for normalization purposes. The absorbance values obtained for the non-phosphorylated target can be used to normalize the absorbance values for the phosphorylated target.