Isoelectric focusing of the chick eye ciliary neuronotrophic factor

A procedure is presented in which a crude extract from selected chick embryo intraocular tissues is submitted to analytical polyacrylamide slab gel isoelectric focusing. The extract contains a protein, ciliary neuronotrophic factor (CNTF) which can be eluted in active form from focused gels in a region occupied by only two protein bands. A "slot" technique is presented in which we demonstrate that the eluted CNTF activity focuses in the very restricted region between the two visible bands and is not associated with either band. Silver stain-densitometry is used to correlate staining intensity with protein concentration and from such an analysis it is concluded that the CNTF protein represents an extremely low proportion of total extract protein and that the minimum CNTF specific activity eluted from gel slices is 10(6) trophic units per mg protein. This one-step procedure will be used in the future to prepare highly purified CNTF for antibody generation.