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The Western Blot is a technique used as a routine in many laboratories to identify the amount of a particular protein in a complex protein sample. Briefly, the technique is based on the separation of proteins by polyacrylamide gel electrophoresis, the transfer of proteins from the gel to a membrane, and the detection of immobilized proteins on the membrane using antibodies.
An example of the application of this technique is the analysis of protein expression in the different cellular compartments (cytoplasm, nucleus, and other organelles).
To do this, what is known as “subcellular fractionation” is carried out, which briefly consists of isolating the proteins and other molecules contained in the different cellular compartments. To further elaborate, for this fractionation different buffers with different compositions in salts and other reagents are used, whose ultimate goal is to break the barriers of both the cell itself (plasma membrane), and the cell organelles and the nucleus (nuclear membrane), exposing its contents.
Thus, cycles of incubation on ice with the buffer are alternated with cycles of centrifugation (400G, 10 min, 4ºC) (Figure 1), obtaining as a result the isolation of the different subcellular fractions (A=cytoplasmic fraction, B=fraction of organelles, C=nuclear proteins), which will be used for Western Blot.
Subcellular fractionation makes it possible to analyse the expression of proteins in the different compartments of the cell by means of Western Blot, and to do this, in addition to the appropriate reagents, cycles of centrifugation are required. During centrifugation, it is necessary to prevent the increase in the temperature of the sample, so a refrigerated centrifuge is necessary to ensure the stability of the temperature of the sample. The microcentrifuge Biocen 22 R has a precooling program with rotor spinning and temperature selectable, guarantees the temperature during the centrifugation process and for greater safety of the sample, remains the refrigeration after the centrifugation process.