The assay is a solid-phase sandwich enzyme-linked immunosorbent assay (ELISA). Standard dilutions and samples are added to the wells precoated with Anti-Bcl-2 antibody (regardless of phosphorylation state)and incubated 2 hours at RT. Bcl-2 binds to the capture antibody. A detection antibody specific for the non-phosphorylated or phosphorylated protein isadded and incubated 1 hr at RT, which results in binding to the immobilized IR protein. An anti-rabbit IgG-HRP binds to the immobilized protein completing the four-member sandwich. The reaction is visualized by tetramethylbenzidine (TMB) substrate, followed by the stop solution. The intensity of the yellow color, measured in a multiwell plate reader at 450 nm, is directly proportional to the concentration of Bcl-2 in standards and samples. The unknown concentrations are calculated from the standard curve run with each assay.
Bcl-2 ELISA is designed to detect and quantify the level of human Bcl-2 protein in cell lysates. Human Bcl-2, the mammalian homolog of the Caenorhabditis elegans protein CED-9, is a cellular oncoprotein with MW 26 kDa, which localizes to the mitochondrial outer membrane, the endoplasmic reticulum, and the nuclear membrane. Bcl-2 has two functions in the cell: to promote cell survival, and to encourage cell cycle exit and quiescence.
phospho-Bcl-2 (pSer70) ELISA detects and quantifies the level of human Bcl-2 protein, phosphorylated on serine 70. No cross-reactivity with mouse and rat cells was observed. For normalizing the content of phospho-Bcl-2 (pSer70) in the samples, use Sigma Bcl-2 (Total) ELISA, which is independent of phosphorylation status (Product Code CS0520).
Features and Benefits
Bcl-2 ELISA is a fast (total time 4 hours), sensitive, and specific alternative to immunoblotting and RIA. The ease of run is facilitated by precoated plates and ready-to-use reagents.
The analytical sensitivity is <0.5 units/mL, which is ~8x more than immunoblotting. Linear regression yielded a correlation coefficient of 0.99. The specificity showed that only the phosphopeptide containing phosphorylated serine 70 blocks the ELISA signal.