Sample preparation for HPLC by Centricon ultrafiltration.
Peptides and other bioactive materials can be purified from complex biological sources by reverse-phase high-performance liquid chromatography (RP-HPLC), provided the mixture is suitably prepared before injection onto an HPLC system. Ultrafiltration offers a convenient and rapid sample preparation technique with numerous advantages over alternative methods such as conventional gel filtration chromatography. We demonstrate the use of ultrafiltration as an HPLC sample preparation step in the purification of peptides bound to class I major histocompatibility complex (MHC-I) membrane proteins. When ultrafiltration was performed with a Centricon-10 ultrafiltration device, peptides were efficiently separated from the alpha (45 kDa) and beta 2m (12 kDa) chains of MHC-I proteins and could be subjected to HPLC without further treatment. Furthermore, even samples as crude as whole cell lysates or supernatants could be prepared for HPLC in a single ultrafiltration step, affording a remarkably straightforward route to the purification of biologically important peptides.
Tsomides
T J
TJ
Center for Cancer Research, Massachusetts Institute of Technology, Cambridge, MA 02139.
D001709:Biotechnology; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D005069:Evaluation Studies as Topic; D015789:HLA-A2 Antigen / Q000302:isolation & purification; D015793:HLA-B7 Antigen / Q000302:isolation & purification; D006801:Humans; D010455:Peptides / Q000302:isolation & purification*; D014462:Ultrafiltration / Q000379:methods*; D001613:beta 2-Microglobulin / Q000302:isolation & purification
D015789:HLA-A2 Antigen; D015793:HLA-B7 Antigen; D010455:Peptides; D001613:beta 2-Microglobulin