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Updated: Sep 21, 2026

Protein Purification-free Method of Binding Affinity Determination by Microscale Thermophoresis
Published on: August 15, 2013
Modulation of nonspecific binding in ultrafiltration protein binding studies
Kyoung-Jin Lee1, Rachel Mower, Tom Hollenbeck
1DMPK, Lion Bioscience, San Diego, California 92121, USA.
Purpose:
The aim of this study was to reduce or prevent nonspecific binding (NSB) of compounds to ultrafiltration (UF) protein binding (PB) testing units.
Methods:
UF units (regenerated cellulose, MWCO 10K) were used for PB and NSB measurements with or without pretreatment with 5% tween 80 (TW 80) or 5% benzalkonium chloride (BAK) on the filter membrane. Dosing solutions (10 microM) in human serum and pH 7.4 phosphate-buffered saline were centrifuged at 3,000 g and room temperature after 1-h incubation in UF testing units. In parallel, a 96-well equilibrium dialyzer was used for PB and NSB measurements in equilibrium dialysis (ED) at 37 degrees C for 4 h. Samples of UF and ED were analyzed by LC/MS or LSC.
Results:
Severe NSB was observed for etoposide, hydrocortisone, propranolol, and vinblastine in UF. In contrast, TW 80 or BAK pre-treatment on the filter membrane decreased the NSB from 87-95% to 13-64% without causing a significant change in membrane integrity. When NSB was below 50% as a result of pretreating agents, PB data of marker compounds were comparable to those of ED.
Conclusions:
The pretreated membrane with TW 80 or BAK showed significantly less NSB for compounds that had a tendency toward high membrane binding. A modified UF method with pretreatment improved the performance of UF and was able to produce comparable PB results to ED.
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