20.500.12556/RUL-146106 Rapid, direct, noninvasive method to determine the amount of immobilized protein Protein immobilization is of utmost importance in many areas, where various proteins are used for selective detection of target compounds. Despite the importance given to determine the amount of immobilized protein, there is no simple method that allows direct, noninvasive detection. In this work, a method based on pH transition, occurring during change of solution ionic strength, was developed. The method utilized the ionic character of the immobilized protein while implementing biologically compatible buffers. Five different proteins, namely, glucose oxidase, horseradish peroxidase, bovine serum albumin, lysozyme, and protein A, were immobilized in different amounts on a porous polymeric matrix, and their pH transition was measured using lactate buffer of various concentrations and pH values. A linear correlation was found between the amount of immobilized protein and the amplitude of the pH transition, allowing the detection down to 2 nmol of immobilized protein. By changing the buffer concentration and pH, the sensitivity of the method could be tailored. Criteria based on the symmetry of the pH transition peak have been developed to determine if a particular measurement is within a linear range. In addition, a mathematical model was developed enabling prediction of pH transition profiles based solely on the protein amino acid sequence, the buffer pK$_a$ value(s), and the amount of immobilized protein. Hence, it can be used to design pH transition method experiments to achieve the required sensitivity for a target sample. Since the proposed method is noninvasive, it can be routinely applied during optimization of the immobilization protocol, for quality control, and also as an in-process monitoring tool. immobilization pH transition protein quantity monomers peptides proteins theoretical chemistry computational chemistry pH imobilizacija pH prehod količina proteina true false true Angleški jezik Slovenski jezik Članek v reviji 2023-05-19 12:10:44 2023-05-19 12:10:44 2023-06-24 04:01:38 0000-00-00 00:00:00 2023 0 0 Str. 5643-5651 iss. 13 Vol. 95 4 Apr. 2023 0000-00-00 Zaloznikova Objavljeno NiDoloceno 0000-00-00 0000-00-00 0000-00-00 66.098 0003-2700 10.1021/acs.analchem.2c05402 147263491 acs.analchem.2c05402.pdf acs.analchem.2c05402.pdf 1 FA89761BB358D1BAC59AF74EE80D6F0D 847546973f73b70d91ea4c3fe6b12b09298c7b64202892cc5a0b19a3481b6616 5e167b51-11c7-11ee-a314-00155dcfd717 https://repozitorij.uni-lj.si/Dokument.php?lang=slv&id=170545 https://pubs.acs.org/doi/10.1021/acs.analchem.2c05402 1 26d90250-f62d-11ed-a314-00155dcfd717 https://repozitorij.uni-lj.si/Dokument.php?lang=slv&id=169225 Fakulteta za kemijo in kemijsko tehnologijo 0 0 0