Validation of primary antibodies specificity in mouse liver and heart mitochondrial protein by western blot after SDS- and Blue Native -PAGE

My experience is based on testing the reliability and functionality of primary antibodies on samples from mouse heart and liver as well as from cells, specifically fibroblasts. The experiment is divided in two main parts. The first part consists in the extraction of mitochondrial proteins from tissues and cells and is followed by the second part, based on the performing\\ of a Western Blot protocol in order to detect some specific mitochondrial proteins by the utilization of primary antibodies in strict and mild conditions, the goal is to determine the functionality of these antibodies. Critical parts of the experience are represented by several steps of the western blotting: separation of proteins by SDS Page (Strict conditions) or Blue Native page (Mild conditions with maintenance of protein complexes), transfer of proteins on membranes, incubation with primary and secondary antibodies and finally the visualization of chemiluminescence. The correct execution of the process is vital for the successful outcome of the experiment.