Analysis of mitogenic activity of proteins after separation by gel electrophoresis

We have used a combination of gel electrophoresis and a cell culture assay in microplates to analyse mitogenic activity in tissue extracts. The proced ure is a modification of the method described by Kuo et al.. The proteins w ere separated by native gel electrophoresis or isoelectric focusing. The ge l was sliced and defined pieces were transferred into tissue culture insert s fitting in 96 well microplates, which contained the test cells. The prote ins diffused from the gel slices directly into the culture supernatant and the mitogenic effects were evaluated by a colorimetric assay (MTT or phosph atase activity). Human interleukin 2 was used to demonstrate the feasibilit y of the method by evaluating the mitogenic effect on the cell line CTLL-2. Extracts of bovine pituitary glands were separated by native gel electroph oresis and isoelectric focusing and several protein bands could be identifi ed which showed a distinct mitogenic effect on human endothelial cells. The method is very sensitive and allows rapid screening of protein mixtures fo r bioactive fractions.