The E.coli cells with recombinant protein were harvested using the lysis buffer. I wanted to store the cells at -80 deg C for future lysis and protein purification. My main concern was that the flash-freeze method may damage the cell itself and may lead to lower protein yield. Should I just store my harvested cells (suspended in lysis buffer) at -80 deg C or should I flash-freeze them first and then store them at -80 deg C?
We store our bacterial pellets at -80 deg for months with no major decrease in protein yields, no need to flash freeze.
what do you mean harvesting or resuspending in Lysis buffer? l would do harvest them from medium,resuspend using water or simple buffer,spin again,throw the sup and store them in -80 or even -20.
Lysis buffer has high amount of salt to facilitate cell lysis when I proceed for protein purification stage. Thanks for the answers.
Ya I know what lysis buffer does, but your goal is to store the cells(unlysed), unless your buffer doesnt have any chemical agent to lyse the cells and to require physical mehtods such as sonification/cell distrupter, you can resuspend in that kind of buffer. Else think about using simple phosphate/tris buffer or water.
Hello,
If you want to store cells without damage, you must remove lysis buffer used for harvesting. Then, flash-freeze or just throwing pellet at -80 will not change dramatically the yield of your further purification.
We routinly pellet our bacteria after induction for a few hours and store them at -80oC with no ill effects on the recombinant protein. Flash freeze with dry ice methanol bath with no liqiud nitrogen available
Hi!
In lysis buffer you actually store cell lysates. If lysates flash-freezed no harm to your recombinant protein will be done. After thaw you can see that your lysate is highly viscous due to the presence of genomic DNA.
You can also obtain the pellet of your cells and freeze them.
If your protein are in the inclusion bodies you can obtain the cell pellet and just put them at -20C in the refrigerator. Your protein will be safe.
Another important point is that flash-freezed cells, cell lysates or other protein preparations should be thawed very slowly on ice. The quick thaw - that is the major source of protein damage.
You can store your bacteria, after eliminating the growth medium, directly at -80ºC for months. At the time of thawing add the lysis buffer with the protease inhibitors needed.
Hey everyone,
Thank you for the valuable advice. I will apply them in my research for sure.
Best,
Sagar
Al already mentioned, you can keep your E. coli cells for months at -80. Even more, the freeze thaw cycle helps (after defrosting) to break your cells, especially if you are using pLys strains.
Make aliquots that you can use up at once, remove the bacteria and freeze them. We had no problems storing bacteria for several month like this in the -80°C freezer and use them afterwards.
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