Background: I am currently over expressing Maltose Binding Protein from BL21(DE3) strain of E.coli. For purification I pass it through maltose resin affinity column. Before that I add cell lysis buffer, sonicate it, centrifuge it to remove the precipitate and filter the supernatant with protein.
Problem: The procedure is long, and I go short in time on one day. Can I do all the procedure up to filtering the supernantant before the day of purification and keep the protein in cell lysis buffer over night for chromatography the next day?
Does it affect the protein in any way ?
If I understand you, you suspend the E. Coli in lysis buffer, sonicate, centrifuge, remove precipitate and then filter the supernatant by pouring through a maltose resin affinity column.
The steps prior to the column take up some time, so you are wondering if you can take the sonicated/lysed bacteria and store them overnight. Have I got that right?
I'm wondering why you can't pour the supernatant into the column, wash it once and store it on the column overnight.
HOWEVER...If I HAD to store lysed and sonicated E. Coli overnight, and I did not want to risk damaging the protein of interest, I'd add protease inhibitors and phosphotase inhibitors to the solution (you might already have those added) and then I would store the lysed/sonicated cells at -80 C. You are after the Maltose Binding Protein and you want to keep them from being damaged. So, stopping all enzyme activity is important. I think at -80, with the inhibitors present, you'd accomplish that...
Keep in mind, I've never stored lysate over night but if I HAD to, that's how I would do it. I have, on the other hand, flash frozen E. Coli in liquid Nitrogen the day before. I flash freeze the bacterial cell pellet and store it at - 80. The NEXT day I use lysis buffer and sonicate. Flash freezing the cells opens them up, so lysing and sonicating the cells can take less time but still be completely effective...
Good luck!
Hi Amar,
I agree with what Tim suggested you. Just a couple of extra things to keep in mind:
- freeze and thaw may damage the protein by causing denaturation
- your protein can be more or less sensitive to proteolysis
this means that you will discover with practice what your protein can or can't tolerate. That said, MBP is a quite well known protein and it is also used as tag to solubilize other proteins. So I think it might be quite resistant and you can probably find information about that in the literature.
As a general rule of thumb, if you run short of time it's always better to freeze the bacterial pellet and start lysis the day after. I keep pellet stored at -20 for several days and never faced particular troubles.
Good luck with your purification!
Nope, as soon as the clarified supernatant is obtained it start to precipitate (GE Healthcare:strategies for protein purification handbook) and will require clarification again the next day through a 0.45 micron filter to remove debris. So, it is better to store the cell pellet as Patrizia suggested and perform both the lysate preparation and purification on the same day.
I agree with the comments given above. It is good to prepare the cell lysates on the same day when you will perform your purification. Depending on the solubility and stability of your protein in your lysis buffer, you can store clarified cell lysates overnight at 4 degree. I did this many times for my GST-tagged proteins. I did not come across any problem. However, each individual protein has its own unique physicochemical properties. Thus, there is no a universal protocol that works for all proteins. Yours may be fine if it is stored overnight at 4 degree. You can try it once and observe the results.
Good luck!
I use to store cells in lysis buffer at -80 C and continue with lysis next day.
Hi there,
If you start your day with frozen cells then thawing/resuspension, sonication, centrifugation and column purification may easily be run on the same day. And therefore you avoid leaving a crude extract overnight (that's what we routinely do in the lab...).
I agree with Dominique Liger sir. Its always better to store cells before lysis n if you manage time, within one day you can do lysis and purification.
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