High resolution preparative isoelectric focusing in layers of granulated gels

Publisher Website
Google Scholar
Abstract
Discontinuous isoelectric focusing in layers of granulated gels is used to separate up to 1000 mg protein with a resolution of 0.01‐0.015 pI. High resolution is achieved with (i) gel layers of reduced thickness (1 mm), (ii) high field strength in the final stage of focusing (100‐200 V/cm), (iii) long separation distances, and (iv) high Volthour (Vh) products ensuring steady state conditions. We describe approaches to rapid focusing based on either short separation distances or extensive prefocusing with resultant short residence times of the separated substances in the gel. The optimum load capacity is 3‐5 mg protein/ml gel‐bed volume while 10 mg protein/ml gle‐bed volume are still well tolerated. At high loading, major components are seen in the gel as transulcent zones which can be easily identified and harvested. Best results are obtained with Sephadex G‐200 (Superfine) or Bio‐Gel P‐60 (minus 400 mesh). Protein visualization with a new printing technique empolying cellulose acetate membranes requires only 2‐3 min and is insensitive to protein overloading and adhering gel particles. A simple and efficient method is described for the electrophoretic removal of carrier ampholytes from the focused proteins. High resolution preparative isoelectric focusing should prove attractive for many applications due to its great flexibility with respect to sample size, gel volume, separation time, protein recovery, ease of operation and reasonable price.