ISSN:
0173-0835
Schlagwort(e):
Capillary electrophoresis
;
Proinsulin conversion
;
Intermediate forms
;
Culture media purification
;
Chemistry
;
Biochemistry and Biotechnology
Quelle:
Wiley InterScience Backfile Collection 1832-2000
Thema:
Biologie
,
Chemie und Pharmazie
Notizen:
Capillary electrophoresis (CE) for the simultaneous and precise quantification of human insulin (hI), proinsulin (hPI) and intermediate forms (des 31, 32; split 65-66 and des 64, 65), released in culture media by engineered cells, is described. Analytical conditions for standard proteins were optimized using a bare silica capillary (20 cm × 50 μm internal diameter). Proteins were monitored at 200 nm and separated at constant voltage. Culture supernatants (12-24 mL) were purified on Sep-Pak Vac C18 cartridges, recovered in 1 mL of acetonitrile:trifluoracetic acid mixture (60:40, v:v), concentrated, ultrafiltered and injected into CE. Protein recovery was 85 ± 14% (n = 5, mean ± standard deviation) with a sensitivity limit of 0.5 nmol/L in the culture media, corresponding to 2 fmol injected in 22 nL. Using the CE method, it was possible to detect and quantify, with precision and accuracy, the release of hPI, hI and intermediate forms directly in the cell culture media, and to compare the proteic pattern released from engineered cells transduced with different hPI gene constructs.
Zusätzliches Material:
2 Ill.
Materialart:
Digitale Medien
URL:
http://dx.doi.org/10.1002/elps.1150190843
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