Evaluation of the Electroosmotic Medium Pump System for Preparative Disk Gel Electrophoresis

• Published in: Analytical biochemistry Vol. 288; no. 2; pp. 168 - 175
• Main Authors: Hayakawa, Mitsuo, Hosogi, Yumiko, Takiguchi, Hisashi, Saito, Shigeno, Shiroza, Teruaki, Shibata, Yasuko, Hiratsuka, Koichi, Kiyama-Kishikawa, Michiko, Abiko, Yoshimitsu
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 15.01.2001
• Subjects: Animals; Dipeptidyl Peptidase 4 - isolation & purification; dipeptidyl peptidase IV; electroosmosis; Electrophoresis, Polyacrylamide Gel - instrumentation; insoluble membrane protein; Kidney - enzymology; Osmosis; preparative gel electrophoresis; Rats; dipeptidyl peptidase IV; preparative gel electrophoresis; electroosmosis; insoluble membrane protein
• ISSN: 0003-2697; 1096-0309
• DOI: 10.1006/abio.2000.4901

This paper describes an improved electroosmotic elution system for preparative sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) based on the epochal idea of H. V. Tan et al. (Nucleic Acids Res. 1988, 16, 1921–1930). In this elution system, a semipermeable membrane, mounted under the gel terminal end, works as the elution pump as well as the partition of the elution chamber. We refer to this system as the “electroosmotic medium pump system.” Operation of the constructed apparatus (3.6 cm i.d. disk gel column) and resolution of the protein bands were examined by separation of the model protein mixture (bovine serum albumin (BSA), ovalbumin, bovine carbonic anhydrase, soybean trypsin inhibitor) and purification of the membrane protein, dipeptidyl peptidase IV (DPP IV). The Spectra/Por 7 dialysis membrane provided a better flow profile for the elution buffer. The four model proteins of the protein mixture were able to be completely separated from each other and recovered without dilution. The maximum protein concentration of eluate achieved was 93 mg/ml, when applying a single component, BSA fraction V, as a sample. Furthermore, the multifunctional ectoenzyme, DPP IV, was purified in a single step.