The 200ml cultures from the last protocol were aliquoted into 8, 50ml centrifuge tubes and spun down at 3000g to separate the cells containing my protein of interest from the media in which they were cultured- the pellet consisted of a layer of bright green (my protein) and a layer of light brown (cellular debris). The supernatant was then removed and the pellet frozen down in a -80*c freezer. Later, the pellet was resuspended in pH and salt specific Tris protein buffer with added DNAase.
This marks the part of the protocol that requires protein extraction from the cells by lysis – the method of choice was several freeze-thaw cycles in liquid nitrogen (6 to be exact). Multiple drastic change in temperature within a short space of time causes a reducing in cellular integrity and cause the cells to lyse, releasing their contents including my protein of interest: His-GFP-NT. At this stage, the sample was spun down at 3000g to pellet out cellular debris.
The first two photos above show the sample during each stage of the freeze-thaw cycles. The latter shows the cellular debris pellet after centrifugation, the green supernatant contains my target protein.
The supernatant was filtered and frozen down, ready for the next part of the protocol – protein purification.
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