Hello everyone,
I am currently working on expressing a protein in Rosetta 2. After harvesting and freezing the cells, the next step is to purify the sample. The initial step involves resuspending the cells in Lysis buffer containing Lysozyme, DNase I, PMSF, and a protease inhibitor.
I have a couple of questions:
Thank you for your assistance.
With lysis buffers you can generally have up to a 1:3 v/v of pellet:buffer. If dealing with small pellets just vortex at a high speed to resuspend. With large pellets you can use a sonic cleaner although you will want to avoid over processing your sample.
Dear Robert Adolf Brinzer
In my experience, the answer to your questions depends from the kind of cell lysis approach that you owuld like to apply after resuspension.
For example in my experience if you are using sonication, cell resuspension by vortexing in 10ml of buffer for gram of wet biomass it is work fine since the sonication itself is able to resuspend and disgregate aggregates that could be not resuspended well by vortex. On the contrary if you are using just enzimatic or chemical lysis is it better to increase the volume of the buffer (eg-20-30ml for each gram) and you have to pay more attention to resuspend well so vortex could be followed by pipetting or agitation during the incubation.
you can find some information about different cell lisis approach at the following link:
https://proteocool.blogspot.com/2021/08/tips-of-week11-
chemicalenzymatic-cell.html
best regard's
Manuele
For lysis buffer volume, a general guideline is to use approximately 5 to 10 mL per gram of wet cell pellet, adjusting based on the density of the pellet and the specific requirements of your purification process. Instead of a brush, utilize a pre-chilled spatula or wide-bore pipette tip for initial resuspension, followed by gentle pipetting or agitation with a rotator in a cold room for a more homogeneous suspension, minimizing cell loss and contamination, and if necessary, brief sonication to disperse any remaining clumps. Again, it depends on the lysis approach.
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