Rapid Detection of Femtogram Amounts of Protein by Gel-Free Immunoblot
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Bulletin of Experimental Biology and Medicine, Vol. 169, No. 6, October, 2020 METHODS
Rapid Detection of Femtogram Amounts of Protein by Gel-Free Immunoblot Y. M. Shlyapnikov, E. A. Malakhova, and E. A. Shlyapnikova
Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 169, No. 6, pp. 788-792, June, 2020 Original article submitted February 28, 2020 The article presents a new method of immunoblotting for simple, rapid, and highly sensitive detection of proteins. Electrophoretic separation of sample is carried out under non-denaturing conditions in a thin conductive layer between cellulose membranes without polyacrylamide gel. The membrane surface is preliminarily modified with azidophenyl groups to photochemically immobilize proteins in situ. For visualization of protein bands, the membranes are treated with magnetic beads coated with specific antibodies, unbound particles are then removed with a magnet. The detection limit in the model system with biotinylated BSA and magnetic beads coated with streptavidin reaches 10 fg or about 105 molecules, while the total blotting time does not exceed 5 min. The method was applied for detection of IgA in a sample of human exhaled air. The method can be used for the analysis of various complex biological samples containing low amounts of the analyte. Key Words: immunoblotting; electrophoresis; magnetic beads; immunoglobulin A Western blotting (WB) is widely used in medicine and molecular biology for detection of proteins with high specificity in mixtures of complex composition. However, the analysis is laborious and time-consuming (usually takes 2-3 days) and depends on the quality of antibodies, which limits its use for rapid detection of low amounts of analyte. Great recent efforts were focused on automation and scaling of WB [2,10,12] and improvement of electrophoretic separation technique and signal detection methods [5]. A significant achievement was the use of photochemical immobilization of proteins [1]. We previously showed that signal detection with magnetic beads increases the sensitivity of the immunoblot by two orders of magnitude in comparison with widely used fluorescent and chemiluminescent detection [9]. To this end, electrophoretic separation of the sample is carried out in a narrow gap between the membranes, followed by in situ immobilization of the analyte on the membrane surface and scanning its Institute of Theoretical and Experimental Biophysics, Russian Academy of Sciences, Pushchino, Moscow region, Russia. Address for correspondence: [email protected]. Y. M. Shlyapnikov
surface with magnetic beads in a flow cell of a special design. The key disadvantage of this method is technical complexity of manufacturing a flow cell, which limits the applicability of this method to a wide range of researchers. The aim of this work was to develop a simple and quick technique to identify femtogram amounts of proteins in a mixture of complex composition that does not require complex equipment and is suitable for use in a conventional b
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