Sensitive Luminometric Method for Protein Quantification in Bacterial Cell Lysate Based on Particle Adsorption and Dissociation of Chelated Europium

A sensitive and rapid assay for the quantification of proteins, based on sample protein adsorption to Eu³⁺-chelate-labeled nanoparticles, was developed. The lanthanide ion of the surface-conjugated Eu³⁺ chelate is dissociated at a low pH, decreasing the luminescence signal. The increased concentration of the sample protein prevents dissociation of the chelate, leading to a high luminescence signal due to the nanoparticle-bound protein. The assay sensitivity for the quantification of proteins was 130 pg for bovine serum albumin (BSA), which is an improvement of nearly 100-fold from the most sensitive commercial methods. The average coefficient of variation for the assay of BSA was 8%. The protein-to-protein variability was sufficiently low; the signal values varied within a 28% coefficient of variation for nine different proteins. The developed method is relatively insensitive to the presence of contaminants, such as nonionic detergents commonly found in biological samples. The existing methods tested for the total protein quantification failed to measure protein concentration in the presence of bacterial cell lysate. The developed method quantified protein also in samples containing insoluble cell components reducing the need for additional centrifugal assay steps and making the concept highly attractive for routine laboratory work

GTP-Specific Fab Fragment-Based GTPase Activity Assay

GTPases are central cellular signaling proteins, which cycle between a GDP-bound inactive and a GTP-bound active conformation in a controlled manner. Ras GTPases are frequently mutated in cancer and so far only few experimental inhibitors exist. The most common methods for monitoring GTP hydrolysis rely on luminescent GDP- or GTP-analogs. In this study, the first GTP-specific Fab fragment and its application are described. We selected Fab fragments using the phage display technology. Six Fab fragments were found against 2′/3′-GTP-biotin and 8-GTP-biotin. Selected antibody fragments allowed specific detection of endogenous, free GTP. The most potent Fab fragment (2A4^GTP) showed over 100-fold GTP-specificity over GDP, ATP, or CTP and was used to develop a heterogeneous time-resolved luminescence based assay for the monitoring of GTP concentration. The method allows studying the GEF dependent H-Ras activation (GTP binding) and GAP-catalyzed H-Ras deactivation (GTP hydrolysis) at nanomolar protein concentrations