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EnglishA liquid chromatographic stationary phase containing immobilized membranes from cells expressing A2A adenosine receptor (A2AAR) is firstly described. Cellular membranes from CHO cells stably transfected with human A2AAR vector (A2A(+)) and from the same cell line transfected with the corresponding empty vector (A2A(−)) were entrapped on immobilized artificial membrane (IAM) support and packed into 6.6 mm I.D. glass columns to create A2A(+)-IAM and A2A(−)-IAM stationary phases. Frontal chromatography experiments on both A2A(+)-IAM and A2A(−)-IAM columns demonstrated the presence of a low specific interaction with the receptor. However, immobilized A2A retained its ability to specifically bind known ligands as demonstrated by the agreement of the calculated K d values with two different chromatographic protocols in comparison to previously reported data. In order to maximize the specific interaction, the same cellular membranes were immobilized on the inner surface of a silica capillary (40 cm × 100 μm I.D.) by non-covalent interactions using the avidin–biotin coupling system to create two open tubular columns A2A(+)-OT and A2A(−)-OT. The open tubular system was characterized by ranking experiments for affinity studies in mixture useful for the selection of new potential candidates.
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| Titolo: | Development of new chromatographic tools based on Adenosine A2A subtype receptor for ligand characterization and screening by FAC-MS | |
| Autori: | ||
| Data di pubblicazione: | 2013 | |
| Rivista: | ||
| Abstract: | A liquid chromatographic stationary phase containing immobilized membranes from cells expressing A2A adenosine receptor (A2AAR) is firstly described. Cellular membranes from CHO cells stably transfected with human A2AAR vector (A2A(+)) and from the same cell line transfected with the corresponding empty vector (A2A(−)) were entrapped on immobilized artificial membrane (IAM) support and packed into 6.6 mm I.D. glass columns to create A2A(+)-IAM and A2A(−)-IAM stationary phases. Frontal chromatography experiments on both A2A(+)-IAM and A2A(−)-IAM columns demonstrated the presence of a low specific interaction with the receptor. However, immobilized A2A retained its ability to specifically bind known ligands as demonstrated by the agreement of the calculated K d values with two different chromatographic protocols in comparison to previously reported data. In order to maximize the specific interaction, the same cellular membranes were immobilized on the inner surface of a silica capillary (40 cm × 100 μm I.D.) by non-covalent interactions using the avidin–biotin coupling system to create two open tubular columns A2A(+)-OT and A2A(−)-OT. The open tubular system was characterized by ranking experiments for affinity studies in mixture useful for the selection of new potential candidates. | |
| Handle: | http://hdl.handle.net/11581/250145 | |
| Appare nelle tipologie: | Articolo |
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