Practical Protein Chemistry - A. Darbre 1989

Traditional strategy for determining protein structure
Fractionation of soluble peptides
DEAE-cellulose column chromatography

Elution of Peptides from DEAE-Cellulose (diethylaminoethyl cellulose) is performed using an increasing gradient of bicarbonate ion concentration [55] (Table 10.5). At pH 8.0, the C-terminal carboxyl groups and the side-chain carboxyl groups of aspartic and glutamic acids are ionized, whereas the side groups of Arginine and Lysine are protonated. The imidazole ring of Histidine is fully deprotonated, and its N-terminal amino group is 50% deprotonated. Peptides are separated according to their net charge at pH 8.0.

Basic peptides are eluted first, followed by neutral and acidic ones. When the sample is applied in a low Ionic strength solution and subsequently eluted with the same solution, good Separation of basic peptides can be achieved. Such "hydrophobic" interactions with the matrix make it possible to separate structurally related peptides that differ only in neutral Amino Acids.

Peptide separation is monitored by measuring the absorbance of the eluate at 280 and 230 nm and using analytical paper Electrophoresis. Based on the data obtained, the fractions are pooled and evaporated to dryness on a rotary evaporator.