Exploration of some proteomic techniques: Western blot, dot blot and two-dimensional electrophoresis, complemented by mass spectrometry
Keywords:
Protein, Western blot, Dot blot, Two-dimensional electrophoresis, Immunodetection, semiquantitativeAbstract
The development of proteomics in biomedicine is of great importance because proteins perform most cellular functions, making them essential molecules in research to understand their biological role. To do this, it is necessary to consider both their biochemical and electrophysical properties in order to achieve their separation and determination. To study the expression of proteins, techniques such as Western blot (WB), dot blot (DB) and two-dimensional electrophoresis are used to identify their expression, intensity and important post-translational modifications during biological activity. WB is based on the separation of proteins according to their charge and molecular weight (MW) and their subsequent transfer to a solid membrane so that a specific antibody recognizes the protein of interest. DB does not require electrophoretic separation, but samples are applied directly to a membrane to provide a semi-quantitative estimate of protein concentration. Finally, there is two-dimensional polyacrylamide gel electrophoresis (2D-PAGE), which can simultaneously separate more than 1,500 proteins from a single sample and can also be combined with various dyes and other techniques. It is important to note that these methods also require specific means of identifying the proteins of interest, such as a MW marker, dyes or antibodies coupled to enzymes or fluorophores. The application of proteomic techniques ranges from basic research to the clinic, with the aim of discovering new biomarkers of biomedical interest. The aim of this review is to provide an overview of the rationale behind these techniques.
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