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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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.
Cell resuspension is done manually by pipetting up and down in a pipette or by using a cell homogenizer when the volume is large enough (> 40 ml). Different sizes of probes are available for sonication depending on the volume of cell suspensions (0.1 to 500 ml). For mechanical lysis using beads, different media and sizes are available depending on the type of cells. The beating of beads on the sample is done by manual vortexing or using an automated homogenizer with interchangeable adapters for different tube sizes (2 ml to 50 ml). This latter equipment allows medium throughput parallel sample preparations. For large volumes of cells, fragile proteins and recovery of macromolecular complexes, lysis by shearing is recommended. Different equipments exist for a range of sample volumes (French press, Dounce homogenizer, Microfluidizer, etc).