Further development of an electroosmotic medium pump system for preparative disk gel electrophoresis.
A simple and practical 6.8-cm-diameter (36.30-cm(2) cross-sectional-area) preparative disk gel electrophoresis device, based on the design of M. Hayakawa et al. (Anal. Biochem. 288 (2001) 168), in which the elution buffer is driven by an electroosmotic buffer flow through the membrane into the elution chamber from the anode chamber was constructed. We have found that the dialysis membranes employed provide suitable flow rates for the elution buffer, similar to those of an earlier 3.6-cm-diameter device, resulting in the prevention of excess eluate dilution. The efficiency of this device was demonstrated by the fractionation of a bovine serum albumin (BSA) Cohn V fraction into monomer, dimer, and oligomer components using nondenaturing polyacrylamide gel electrophoresis (native-PAGE). The maximum protein concentration of the eluate achieved was 133 mg/ml of BSA monomer, which required a dilution of the eluate for subsequent analytical PAGE performance. As a practical example, the two-dimensional fractionation of soluble dipeptidyl peptidase IV (sDPP IV) from 50 ml fetal bovine serum (3.20 g protein) per gel is presented. The sDPP IV enzyme protein was recovered in a relatively short time, utilizing a 6.5% T native-PAGE and subsequential sodium dodecyl sulfate-PAGE system. This device enhances the possibility of continuous electrophoretic fractionation of complex protein mixtures on a preparative scale.
Hayakawa
Mitsuo
M
Department of Biochemistry, Nihon University School of Dentistry at Matsudo, Matsudo, Chiba 271-8587, Japan. mitsuoha@mascat.nihon-u.ac.jp
Hosogi
Yumiko
Y
Takiguchi
Hisashi
H
Shiroza
Teruaki
T
Shibata
Yasuko
Y
Hiratsuka
Koichi
K
Kiyama-Kishikawa
Michiko
M
Hamajima
Susumu
S
Abiko
Yoshimitsu
Y
D000818:Animals; D002417:Cattle; D018819:Dipeptidyl Peptidase 4 / Q000097:blood / Q000302:isolation & purification; D004589:Electrophoresis, Disc / Q000295:instrumentation* / Q000379:methods
D018819:Dipeptidyl Peptidase 4