Extraction of endogenous or recombinant proteins and protein complexes from cells or tissues and their recovery in solution for further analysis
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For purification and subsequent research purposes, the protein has to be brought into solution by breaking the tissue or cells containing it. Several methods of cell lysis are commonly used to achieve this : mechanical disruption, sonication using high frequency sound waves, liquid homogenization and freeze/thaw cycles. For example, the beating of glass beads on cells crush cell walls, cell suspensions are sheared by forcing them through a narrow space and high pressure/sudden depressurization disrupt cells. The choice of the lysis method depends on the type of cells (bacteria, yeast, mammalian cells, etc), the volume of cell suspension and the fragility of the proteins to be recovered. The composition of the lysis buffer is also crucial to maintain the proteins of interest in the soluble fraction after cell breakage.
Cell pellets are resuspending in an appropriate lysis buffer supplemented with proteases inhibitors to slow down proteolysis and optionally with nucleases to decrease the viscosity of the sample due to the release of nucleic acid material. Localized heating within a sample can occur with many of the lysis techniques, leading to protein aggregation and denaturation. To avoid this problem, it is essential to pre-chill equipment and keep samples on ice at all times. Depending on the method and the volume of cell suspension, lysis should take about minutes to hours.