Isolation of Intermediate Filaments

Intermediate filaments (IFs) are found in most eukaryotic cells and are made up of various IF proteins. IFs are highly insoluble in conventional extraction buffers and are therefore commonly purified under denaturing condition. Purified IF proteins can be reassembled into filaments by dialysis. At least 65 IF proteins are found in humans, and the procedures for the purification of each subunit vary somewhat, although many basic steps are similar. To illustrate the isolation of IFs, a detailed protocol is described for purifying neurofilament proteins (NFL, NFM, and NFH subunits) from bovine spinal cord. These three proteins form the predominant IF network in mature neurons. An alternative method for the purification of NFL from a prokaryotic expression system is also included. The isolation of recombinant proteins from bacteria is quite straightforward and may therefore be the method of choice for producing and purifying IFs. Finally, there is a discussion of the purification methods of other IF proteins.