The recovery of proteins in polyacrylamide gel electrophoresis (PAGE) has not been previously investigated systematically. The fractionation of hemoglobins A and 8 (1) by which milligram-preparative PAGE (2) was initially introduced yielded nearly quantitative recovery of hemoglobin, a finding which delayed recognition of the fact that severe recovery problems are frequently encountered. Later work in this laboratory (3-5) with several different proteins and buffer systems and the few reports in the literature which gave recovery data (6-S) indicated that protein recoveries were usually of the order of 50-70s. Therefore an attempt was made to set ~11) a preparative PAGE system by which recovery could he quantitatively determined, to investigate the chemical mechanisms responsible for the loss of protein in PAGE, and to evolve pract,ical ways to improve recoveries of protein in preparative PAGE. Recovery and resolving capacity were determined for several types of preparative apparatus.
Methods
(1) Preparative PAGE Apparatus
Preparative PAGE apparatus A is a new design based on the Fractophorator apparatus originated by Schenkein et nl. ( 9) and commercially available from Buchler Instruments, Inc., Fort Lee, N. J. The apparatus was redesigned to give field symmetry, a small elution chamber, temperature control, hydrostatic equilibration of the gel column by immersion in the Lower Buffer reservoir, vertical alignment, and an elution buffer delivery stroke of 1.3 ml/5 min. The apparatus is described in Appendix I (available on request') and depicted schematically in Fig. 1. Other preparative PAGE apparatus used included the Polyprep 200 apparatus (Buchler Instruments) with Upper Buffer reservoir of 2 liter capacity, symmetrical triple elution buffer inlet int,o the elution chamber, and centrally affixed upper platinum electrode. This apparatus is desig-