I want to prepare crude membranes from HEK293freestyle cells. Since I just recently started to work with this cells, I'm not sure what is the best way to crack the cells. I would either use a sonicator or a dounce homogenizator. Anyone with some experience?
Thank you.
I think ultrasonic cell disrupters (sonicator) work much better than blender style (shear) mechanical homogenators but you need to use a focused ultrasonic source. A standard ultrasonic bath is not focused enough to do the job thoroughly. There are also some chemical options like Triton X-100 or DMSO. Depending on the cell contents that you are looking for, you may need to add some other chemicals, whether you use mechanical or chemical means, to prevent digestion or denaturing of the target molecule, e.g. 2-mercaptoethanol is used to prevent RNA digestion by ribonucleases released during cell lysis.
We use hypotonic lysis and it works fine. Because we use attached cells we follow with a cell scraper (" rubber policeman"). My understanding is that you are doing this with suspension cells so I would collect the cells by centrifugation at low speed, lyse the cells in lysis buffer and triturate (draw up and down) with a syringe and 21 ga needle. Then collect the membranes by ultracentrifugation and typically use a 1 ml 60% sucrose cushion which will collect total membranes. This will appear as a white/beige band just above the sucrose cushion. I have attached our lab protocol. Note that this is for our prostaglandin receptor expressing membranes so you will not need to add indomethacin to inhibit prostaglandin synthesis, unless of course you are expressing prostaglandin receptors. Let me know if you have further questions; we do this all of the time, but not on cells in suspension.
We are isolating membrane proteins from HEK 293F and follow one of two simple protocols:
Depending on the protein, sometimes one, the other, or both methods work equally well. Following this we proceed to e.g. an affinity resin step. Hope that helps.
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