title: Method for estimation of protein isoelectric point.
abstract: Adsorption of sample protein to Eu(3+) chelate-labeled nanoparticles is the basis of the developed noncompetitive and homogeneous method for the estimation of the protein isoelectric point (pI). The lanthanide ion of the nanoparticle surface-conjugated Eu(3+) chelate is dissociated at a low pH, therefore decreasing the luminescence signal. A nanoparticle-adsorbed sample protein prevents the dissociation of the chelate, leading to a high luminescence signal. The adsorption efficiency of the sample protein is reduced above the isoelectric point due to the decreased electrostatic attraction between the negatively charged protein and the negatively charged particle. Four proteins with isoelectric points ranging from ~5 to 9 were tested to show the performance of the method. These pI values measured with the developed method were close to the theoretical and experimental literature values. The method is sensitive and requires a low analyte concentration of submilligrams per liter, which is nearly 10000 times lower than the concentration required for the traditional isoelectric focusing. Moreover, the method is significantly faster and simpler than the existing methods, as a ready-to-go assay was prepared for the microtiter plate format. This mix-and-measure concept is a highly attractive alternative for routine laboratory work.
authors.affiliation: Laboratory of Biophysics and Medicity Research Laboratory, University of Turku, Tykistökatu 6A, FI-20520 Turku, Finland. sari.pihlasalo@utu.fi
authors.forename: Sari
authors.initials: S
authors.lastname: Pihlasalo
authors.forename: Laura
authors.initials: L
authors.lastname: Auranen
authors.forename: Pekka
authors.initials: P
authors.lastname: Hänninen
authors.forename: Harri
authors.initials: H
authors.lastname: Härmä
mesh_terms: D000327:Adsorption; D000418:Albumins / Q000032:analysis*; D000818:Animals; D002256:Carbonic Anhydrases / Q000032:analysis* / Q000378:metabolism; D002417:Cattle; D002614:Chelating Agents / Q000737:chemistry; D004912:Erythrocytes / Q000201:enzymology; D005063:Europium / Q000737:chemistry; D005753:Gastric Mucosa / Q000201:enzymology; D057166:High-Throughput Screening Assays; D006657:Histones / Q000032:analysis; D006863:Hydrogen-Ion Concentration; D007526:Isoelectric Point; D053758:Nanoparticles / Q000737:chemistry; D010179:Pancreas / Q000196:embryology; D010434:Pepsin A / Q000032:analysis*; D012710:Serum Albumin, Bovine / Q000737:chemistry; D055672:Static Electricity; D013552:Swine; D013950:Thymus Gland / Q000737:chemistry; D014362:Trypsinogen / Q000032:analysis*; D005719:gamma-Globulins / Q000032:analysis*
chemical_list: D000418:Albumins; D002614:Chelating Agents; D006657:Histones; D005719:gamma-Globulins; D012710:Serum Albumin, Bovine; D005063:Europium; D014362:Trypsinogen; D010434:Pepsin A; D002256:Carbonic Anhydrases