Microplate screening assay for binding of ligands to bovine or reindeer β-lactoglobulins

A microplate screen to estimate metal-binding affinities of metalloproteins

“…tryptophan) upon sequential exposure to a ligand through step-wise titration can generate binding curves for estimating KD, which therefore complements ITC by helping to calculate c. To increase the throughput for more extensive library screening, microplate readers equipped with fluorescence-reading capabilities can be used instead of spectrofluorometers, which has been tested for interactions between albumins and proteases with organic compounds. [3][4][5] Microplate-based ITFQ-LTA (microITFQ-LTA) has also been used to study interactions between metalloproteins and UO2 2+ , but these researchers' decision to use Equation (1), the Stern-Volmer equation (variables described further below), to fit the fluorescence quenching data is questionable. 6,7 (1)…”

Tracking of β-Lactoglobulin Binding Compounds with Biofingerprinting Chromatogram Analysis of Natural Products

“…The recovery of piperine was 82%. The binding of piperine was also confirmed with our miniaturized fluorescence based method [10]. Next, the chromatograms of the P. nigrum extract before and after filtration were compared and it was shown that piperine was highly bound to βLG.…”

“…Representative HPLC chromatograms were recorded at 265, 270 and 340 nm according to UV-vis spectra of the extracts. The confirmation of binding of piperine and rutin to βLG were made using the miniaturised fluorescence based method of Riihimäki et al [10].…”

“…Thus we were interested in evaluating the possible binding of components in tea extracts to bovine βLG, which belongs to the lipocalin protein family and is the main whey protein in bovine milk [6]. The physiological function of βLG is not known, but it could bind hydrophopic ligands like retinol and fatty acids into the central cavity [7][8][9][10]. The retinol binding site in the central cavity has been proved with crystallographic studies [11,12].…”

“…[15][16][17] In this study the binding of compounds in tea extracts from Camellia sinensis (L.) O. Kunze (black tea) and Urtica dioica L. (nettle) was studied. Since our previously miniaturised fluorescence based method [10] for βLG binding is suitable only for pure compounds, we developed a method of ultrafiltration sampling combined with HPLC to study the βLG-binding property of extracts. A similar method of microdialysis sampling combined with HPLC has been previously used in DNA-binding studies with extracts of Chinese medicines [18].…”