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A quantitative western blot technique using TMB: Comparison with the conventional technique
Author(s) -
Ntoukas Aristeidis,
Niarchos Athanasios,
Tsika Aikaterini C.,
Mantzoukas Spiridon,
Spyroulias Georgios A.,
Poulas Konstantinos
Publication year - 2021
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/elps.202000306
Subject(s) - linearity , blot , western blot , lysis , protein detection , signal (programming language) , biological system , protocol (science) , computer science , chemistry , chromatography , analytical chemistry (journal) , nanotechnology , materials science , biology , electronic engineering , biochemistry , engineering , medicine , alternative medicine , pathology , gene , programming language
Numerous molecular biological experiments performed throughout the world require the detection or quantification of a protein of interest. Western blotting is one of the most popular techniques used for this purpose and offers quantitative information with the aid of specialized software. However, its dependence on the picture that is captured, and the background and the absence of a common protocol prevent the technique from being completely quantitative. To overcome these obstacles, we present a simple and reliable assay that is similar to the regular technique, with the exception of the last stage of band visualization and quantification. We propose that small pieces of the blot that include the protein of interest can be cut and dipped in a small volume of 3,3',5,5'‐tetramethylbenzidine solution, giving a colorimetric signal with linear dependence on the quantity of the protein. The reaction is stopped with H 2 SO 4 , and the signal is measured in a plate reader. This modification shows high linearity without additional costs and can be applied for both purified proteins and proteins found in a lysate. The results obtained with our proposed technique were compared with those obtained by the conventional method and proved to be more reliable.