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Frontal Affinity Chromatography for the Screening of Mixtures

[ Vol. 5 , Issue. 5 ]

Author(s):

N. W.C. Chan, D. F. Lewis, S. Hewko, O. Hindsgaul and D. C. Schriemer   Pages 395 - 406 ( 12 )

Abstract:


A protein stationary phase for frontal affinity chromatography was prepared, containing biotinylated β-galactosidase immobilized to controlled pore glass beads via covalently bonded streptavidin. Single microaffinity columns of approximately 30 pmol of active β-galactosidase were prepared from this material and characterized with a known ligand by frontal analysis. These columns were used to measure the specific interactions between the bound β-galactosidase and a library of modified β-galactopyranosides using electrospray mass spectrometry as the means of detection. The library contained 89 entries, each representing 4 diastereomers for a total of 356 library members. A single entry was analysed revealing differential activity among the 4 isomers. The library was grouped into 10 mixtures of 24-40 members each with each mixture infused under frontal chromatographic conditions. This deconvolution procedure led to the identification of 34 entries containing isomers with Kd values better than 10 μM. A method based on a displacement principle was implemented as a rapid prescreen which served as the basis for a parallel column high throughput screening assay.

Keywords:

frontal affinity chromatography

Affiliation:

The University of Calgary,Department of Biochemistry and Molecular Biology, Calgary Alberta,Canada



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