Hello all,
I am trying to isolate and purify my recombinant protein. I usually lyse the cells using sonication (15 sec on, 15 sec off for 7:30 mins; 2X) for all my other recombinant proteins. For this other protein however sonication seems to "precipitate" my protein. I get a whitish looking pellet that contains my protein. I have tried using bugbuster to lyse the cells and I am able to get some protein in the supernatant, although there is still a large amount in the pellet.
Any ideas on a gentle way to release as much of my protein from the cells without precipitation?
Thank you all in advance
My preferred method is to use a soft lysing buffer w/ lysozyme and then freeze-thaw going from liquid nitrogen to a ~42C bath, followed by a few cycles of ultrasonication with medium power, and repeat this cycle 3 times. You can add glycerol as well to help maintain solubility.
Thank you Vincent,
What is a soft lysis buffer? and how cycles of sonication do you recommend?
I do have lysozyme and 10% glycerol in my lysis buffer
Dear Yacoba
is your protein contain cisteines or it has a isoelettric point very close to the buffer that are you using for sonication?
Because in this case you can just try to change a little the buffer that are you using for sonication.
alternativelly one lysys buffer that in my experience work quite well is cell lytyc express of sigma aldrich (c1990) .
you need to resuspend the cell into the buffer..1g of cells in about 20ml of buffer and incubate 30' at rt under agitation.
good luck
Manuele
Here is a generic protocol, you can add a non-ionic detergent (ex: Tween) at low concentration as well but I tend to steer clear of the harsh chemicals like PDMS.
Thank you all for the suggestions. I did consider the possibility of the formation of inclusion bodies, therefore I induce at 18 C for 4 hrs. I have also experimented with different IPTG concentrations .
The reason I think it may be due to the lysis method is because I am able to get some soluble protein when I use BugBuster for lysis (verified by immunoblotting and activity check). I just feel that there is insufficient lysis with BugBuster.
Anymore suggestions are welcome.
Thank you
Dear Yacoba
I think that the Cell lytyc express from Sigma ( cod C1990) may represent a possible alternative. it works every well in my hands.
good luck
Manuele
I prefer to use Geneaid extraction kit for isolation bacterial protein, contain simple and a good results for bacterial protein extraction
- Badges
- Science topic
- Similar topics
- Correction