Alkaline Phosphatase Purification: A Comparison of Prep-PAGE with Cyclic Processes

Semicontinuous protein separation via variable-field-strength electrophoresis

“…As the power increases from 3 to 10 W, the cycle or batch time decreases from 13 to 8 h. The field strengths corresponding to these wattages are 170 and 331 V, resp. Comparison of these runs in Table I shows that the separation factor increases from 6 to 13 when the field strength decreases from 331 to 231 V, but that the separation factor decreases from 13 to 11 when the field strength decreases from 231 to 170 V. Thus, the observed separation goes through a maximum at about 231 V or 5 W. The optimum wattage for maximum separation was also found to be 5 W in our previous experiments on HPAP (Chao et al, 1983), which is reasonable because the leading peak is albumin in both the crude HPAP mixture and the hemoglobin-albumin mixture. A higher field strength would presumably he required in order to obtain sharp peaks and good separation if HPAP or hemoglobin were to be separated from another protein with lower electrophoretic mobility.…”

“…Evidently, the pH of the gel and the feed is more important than the pH of the elution buffer. In our previous experiments on HPAP, an optimum pH for the purification was observed (Chao et al, 1983).…”

“…Crude HPAP is difficult to purify and typifies the separation problems encountered in most biochemical separations. There are six isomeric forms of the enzyme and the crude enzyme mixture contains protein impurities such as human placental albumin with molecular weights and/or isoelectric points close to the values for HPAP, thus making these impurities difficult to remove (Chao et al, 1983). Furthermore, the enzyme concentration is usually measured by recording its activity compared to the total protein concentration in the sample.…”

“…Their data show total protein eluted with respect to fraction number or time. The operating conditions in the present work are based on the batch studies by Righetti and Secchi (1972) and Chao et al (1983). The elution profiles for the individual proteins are shown.…”