An examination of ciliary neuronotrophic factors from avian and rodent tissue extracts using a blot and culture technique.

We have previously reported a technique for determining the apparent molecular weight (Mr) of ciliary neuronotrophic factors (CNTFs) in crude extracts. This method involves SDS-polyacrylamide gel electrophoresis of the extract. Western blotting and culture of purified ciliary ganglion neurons on the paper containing the blotted lane. Neurons will survive only if in direct contact with the trophic factor band and the surviving neurons, when stained with a vital dye, will outline the CNTF band thereby indicating the Mr of the active polypeptide. Here we have modified this 'blot and culture' technique by including Mr standard proteins in the same electrophoretic lane with the samples, identifying the proteins by staining the nitrocellulose blot with Amido black, marking the standard bands with pinholes and destaining the blot prior to seeding neurons onto it. The active CNTF polypeptides can then be identified by their ability to support the 24-h survival of cultured ciliary neurons. This modified technique was used to determine the Mr of CNTF activities in several chick and rat tissue extracts of selected developmental ages and to ascertain if the two forms of CNTF are exclusive to chick and rat, embryonic and adult, or eye and nerve tissues. We report that the above modifications permitted a more accurate method for Mr determination than the previous method, only two apparent forms of CNTF were recognized, the Mr found for each form is 25 kDa and 28 kDa, both forms can be present in chick and rat tissues and from embryonic and adult sources and the 28 kDa form is predominant in rat while the 25-kDa form is predominant in chicken tissues.