Purification of naturally occurring peptides by reversed-phase HPLC.
Reversed-phase high performance liquid chromatography (HPLC) has become the method of choice for the purification of peptides and small proteins (M(r) < 10,000 Da) from natural sources. The technique combines high resolution and recovery with ease and speed of operation and is applicable to a wide range of peptides with different physicochemical properties. This protocol describes procedures for (1) the extraction of a biologically active peptide from animal tissue, (2) concentration of the extracts and partial purification on Sep-Pak cartridges, and (3) purification to near homogeneity on a range of silica-based HPLC columns. Standard operating procedures involve acetonitrile as organic modifier, trifluoroacetic acid as ion-pairing reagent and sequential chromatographies on octadecyl (C18), butyl (C4) and diphenyl wide-pore (300 A) columns under gradient elution conditions. The limiting factor in the time taken to isolate a peptide is usually the speed at which assays to detect the peptide can be performed, but purifications can generally be accomplished within 1 or 2 weeks.
Conlon
J Michael
JM
Department of Biochemistry, Faculty of Medicine and Health Sciences, United Arab Emirates University, PO Box 17666, Al-Ain, United Arab Emirates. jmconlon@uaeu.ac.ae
D000097:Acetonitriles; D000818:Animals; D002845:Chromatography; D002851:Chromatography, High Pressure Liquid / Q000295:instrumentation / Q000379:methods*; D010455:Peptides / Q000302:isolation & purification*; D012822:Silicon Dioxide; D014020:Tissue Extracts / Q000737:chemistry; D014269:Trifluoroacetic Acid
D000097:Acetonitriles; D010455:Peptides; D014020:Tissue Extracts; D012822:Silicon Dioxide; C042953:octadecyl silane-bonded silica; D014269:Trifluoroacetic Acid; C032159:acetonitrile