Extension of separation range in capillary isoelectric focusing for resolving highly basic biomolecules.
The non-availability of commercial carrier ampholytes in the pH range greater than 11 has contributed to difficulties in focusing and resolving highly basic proteins/peptides using capillary isoelectric focusing (cIEF). Two different approaches, involving the use of N,N,N',N'-tetramethylethylenediamine (TEMED) and ampholyte 9-11, are investigated for their effects on the extension of separation range in cIEF. The addition of TEMED into pharmalyte 3-10 not only prevents the peptides/proteins from focusing in sections of the capillary beyond the detection point, but also extends the separation range to at least isoelectric point (pI) 12. The combination of ampholyte 9-11 with pharmalyte 3-10 surprisingly provides baseline resolution between bradykinin (pI 12) and cytochrome c (pI 10.3). The sample mixture, containing bradykinin, the high-pI protein calibration kit (pI 5.2-10.3), and cytochrome c digest, is employed to demonstrate the cIEF separation of proteins and peptides over a wide pH range of 3.7-12.
Mohan
Deepa
D
Department of Chemistry and Biochemistry, College of Life Sciences, University of Maryland, College Park, MD 20742, USA.
Lee
Cheng S
CS
D001920:Bradykinin / Q000302:isolation & purification*; D002021:Buffers; D002138:Calibration; D003574:Cytochrome c Group / Q000302:isolation & purification*; D007525:Isoelectric Focusing / Q000379:methods*; D013056:Spectrophotometry, Ultraviolet
D002021:Buffers; D003574:Cytochrome c Group; D001920:Bradykinin