Method for Estimation
of Protein Isoelectric Point
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Abstract
Adsorption of sample protein to Eu<sup>3+</sup> 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<sup>3+</sup> 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