Continuous flow electrophoretic separation of proteins and cells from mammalian tissues.
A new continuous flow electrophoretic separator for cells and macromolecules was built and tested in laboratory experiments and in the microgravity environment of space flight. Buffer flows upward in a 120-cm long flow chamber, which is 6 cm wide X 1.5 mm thick in the laboratory version and 16 cm wide X 3.0 mm thick in the microgravity version. Electrophoretic subpopulations are collected in 197 fractions spanning 16 cm at the upper end of the chamber. The electrode buffer is recirculated through front and back cooling chambers, which are also electrode chambers. Ovalbumin and rat serum albumin were used as test proteins in resolution and throughout tests; resolution of these two proteins at 25% total w/v concentration in microgravity was the same as that found at 0.2% w/v concentration in the laboratory. Band spreading caused by Poiseuille flow and conductance gaps was evaluated using polystyrene microspheres in microgravity, and these phenomena were quantitatively the same in microgravity as in the laboratory. Rat anterior pituitary cells were separated into subpopulations enriched with cells that secrete specific hormones; growth-hormone-secreting cells were found to have high electrophoretic mobility, whereas prolactin-secreting cells were found to have low electrophoretic mobility. Cultured human embryonic kidney cells were separated into several electrophoretic subfractions that produced different plasminogen activators; a medium-high-mobility subpopulation and a medium-low-mobility subpopulation each produced a different molecular form of urokinase, whereas a high- and an intermediate-mobility subpopulation produced tissue plasminogen activator. Canine pancreatic islets of Langerhans cells were separated into subpopulations, which, after reaggregation into pseudoislets, were found to be enriched with cells that secrete specific hormones; insulin-secreting beta cells were found in lowest mobility fractions, whereas glucagon-secreting alpha cells were found in the highest mobility fractions. Results of particle electrophoresis experiments were comparable in microgravity and in the laboratory, since cell densities that overloaded the carrier buffer (resulting in zone sedimentation) were avoided, and a 500-fold increase in protein throughput was achieved without compromising resolution in microgravity.
D000818:Animals; D002460:Cell Line; D002469:Cell Separation / Q000379:methods*; D004285:Dogs; D004586:Electrophoresis / Q000295:instrumentation / Q000379:methods; D004622:Embryo, Mammalian; D005934:Glucagon / Q000378:metabolism; D013006:Growth Hormone / Q000378:metabolism; D006801:Humans; D007328:Insulin / Q000378:metabolism; D000078790:Insulin Secretion; D007515:Islets of Langerhans / Q000166:cytology / Q000378:metabolism; D007668:Kidney / Q000166:cytology / Q000378:metabolism; D008297:Male; D010903:Pituitary Gland, Anterior / Q000166:cytology / Q000378:metabolism; D010960:Plasminogen Activators / Q000378:metabolism; D011388:Prolactin / Q000378:metabolism; D011506:Proteins / Q000302:isolation & purification*; D051381:Rats
D007328:Insulin; D011506:Proteins; D011388:Prolactin; D013006:Growth Hormone; D005934:Glucagon; D010960:Plasminogen Activators