In flow activation of diol-silica with cyanogen bromide and triethylamine for preparing high-performance affinity chromatographic columns.
A new coupling strategy using pre-packed diol-silica supports to obtain affinity columns for high-performance affinity chromatography (HPAC) is described. These columns were prepared by "in flow" activation in which solutions containing anhydrous solutions of CNBr and triethylamine are separately pumped to a mixer and then onto a pre-packed diol-silica column. Recycling the amino ligand to be coupled several times over the activated silica diol columns results in ligand immobilization. DNA (the Op 1 lac operator), 6-aminohexyl-Cibacron and a peptide (melittin) were all successfully "in flow" coupled to freshly activated columns. Methods for CNBr activation of pre-packed diol-silica column were developed for one, two or three pump HPLC systems. The supports were successfully used for the HPAC purification of a Lac repressor-beta-galactosidase fusion protein, alcohol dehydrogenase, and calmodulin. Columns prepared by in flow activation/coupling procedures were shown to be stable for at least 14 months. Also, in flow activated silica columns could be stored in anhydrous acetone for at least 3 months prior to coupling. Our experiments with these affinity ligand columns (DNA-silica, aminohexyl-Cibacron F3GA-silica, and melittin-silica), suggests that this is a very successful coupling protocol for producing a variety of HPAC columns.
Jurado
Luis A
LA
Department of Biochemistry, College of Medicine, University of Tennessee, 858 Madison Avenue, Suite G01, Memphis, TN 38163, USA.
Jarrett
Harry W
HW
D002846:Chromatography, Affinity / Q000295:instrumentation*; D002851:Chromatography, High Pressure Liquid / Q000295:instrumentation*; D003488:Cyanogen Bromide / Q000737:chemistry*; D005021:Ethylamines / Q000737:chemistry*
D005021:Ethylamines; D003488:Cyanogen Bromide; C016162:triethylamine