The IκBα (Phospho-Ser32) Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect IκBα (Phospho-Ser32) protein expression profile in cells. The kit can be used for measuring the relative amounts of IκBα (Phospho-Ser32) in cultured cells as well as screening for the effects that variuos treatments, inhibitors (ie siRNA or chemicals), or activators have on IκBα (Phospho-Ser32).
This kit contains Crystal Violet Staining normalization protocol. It provides a means to normalize Absorbance 450 nm of primary antibodies to cell density of the cells in the same well. This provides an accurate account for stimulations and cell densities during the assay. By comparing the proportional values obtained from the experimental wells to those of the control wells (e.g. untreated cells), it is possible to determine the extent to which the stimulant(s) have affected the expression levels.
Western blot analysis of lysates from 1) Rat kidney, 2) 3T3, 3) K562, 4)Hela cells, (Green) primary antibody was diluted at 1:1000, 4°over night, Dylight 800 secondary antibody(Immunoway:RS23920)was diluted at 1:10000, 37° 1hour. (Red) Tubulin β Monoclonal Antibody(5G3) (Immunoway:YM3030) antibody was diluted at 1:5000 as loading control, 4° over night,Dylight 680 secondary antibody(Immunoway:RS23710)was diluted at 1:10000, 37° 1hour.