A General Method to Recondition and Reuse Biacore Sensor Chips Fouled with Covalently Immobilized Protein/Peptide

Abstract

Of significance in the routine use of BIAcore is the cost of the sensor chips. This is particularly evident during the phase of method development of an assay where it is not unusual to expend several chips in a day in attempts to optimize immobilization conditions for a novel peptide or protein. In addition, it is accepted practice to discard a chip once its ligand binding capacity has diminished to an unacceptable level. While the high cost of sensor chips has been addressed to some degree through the recent introduction of research-grade sensor chips, we were interested in assessing the possibility of regenerating or reconditioning sensor chips in order to allow them to be reused. In particular, we concerned ourselves with regenerating sensor chips onto which peptide or protein had been immobilized. Our aim was to develop a general procedure that would allow reuse of such chips but would not decrease ligand immobilization capacity or increase nonspecific ligand adsorption properties. We present a method which employs a combination of enzymatic (Pronase E) and chemical (bromoacetic acid) treatments of used sensor chips. Regeneration requires an overnight incubation of the sensor chip ex situ so that one can continue to perform BIAcore experiments. The data demonstrate that this simple two-step procedure substantially removes immobilized proteins such as IgG, Protein G, an HIV-1 envelope glycoprotein (gp120) and a neoglycoprotein based on bovine serum albumin, as determined by reflectance measurements and X-ray photoelectron spectroscopy. In addition, a high density of proteins can be immobilized onto the reconditioned surfaces, since reactive carboxylic acids are regenerated by the procedure.