Beta-Actin is highly conserved proteins and is ubiquitously expressed in all eukaryotic cells at a constant level regardless of experimental treatment or technical procedure in most cases. Therefore, measurement of beta-Actin is generally used as an internal control for experimental error. Signosis has provided b-actin sandwich ELISA assay specifically to detect the endogenous levels of beta-actin, which can be used as internal control for most of ELISA assays with human, mouse and rat samples.
- Simple Procedure - Typical sandwich ELISA procedure.
Normalization - The assay can be used to normalize the loading control in a high-throughput way.
- Quantitative Analysis - Protein standard is provided for quantitative analysis of your samples.
Beta-actin ELISA is based on the principle of a solid phase enzyme-linked immunosorbent assay. The assay utilizes a rabbit polyclonal beta-actin capture antibody for immobilization on the microtiter wells, and a mouse monoclonal beta-actin detection antibody along with anti-mouse conjugated to horseradish peroxidase (HRP) for detection. The test sample is allowed to react simultaneously with the two antibodies, resulting in the beta-actin molecules being sandwiched between the solid phase and enzyme-linked antibodies. After incubation, the wells are washed to remove unbound-labeled antibodies. A HRP substrate, TMB, is added to result in the development of a blue color. The color development is then stopped with the addition of stop solution changing the color to yellow. The concentration of beta-actin is directly proportional to the color intensity of the test sample. Absorbance is measured spectrophotometrically at 450 nm.
Figure 1. Detection of beta-actin protein with beta-actin ELISA kit. HeLa cells were grown in a 12-well plate. Cell lysate was prepared with Cell Lysis Buffer, 1:2 serially diluted with 1 x Diluent buffer and subject to ELISA assay.