I am sonicating DE3 cells for extracting my recombinant protein. I am getting my protein and things are working fine. But, I am curious to know about over or under sonication and overestimation by Bradford method.The crude extract protein which I estimate by Bradford is way higher than what is seen on SDS-PAGE. Also, I am worried about sonication conditions. Can anyone suggest the sonication conditions for E. coli DE3 cells with a 6 mm probe of sonics vibracell vcx130? In my previous lab, I was using sonics 500 with small probe which works fine with 1 ml cell suspensions. I always saw clearing of cell suspension post-sonication, but it is not happening here. I am working with 12 ml volume and have tried different amount of cells.
u can keep amplitude 32 with 10 minutes time period. but time period of 10 min will be in a programmed way in the form of 10 sec on and successively followed by 20 minutes off. It works fine in my case and hope the same for u.
good luck.
Does the target protein form inclusion bodies and is expressed at high level? In that case, you will never get a clear suspension even at 100% cell lysis.
Thanks Ajeet for the conditions. @Alejandro, is turbidity because of unlysed cells/incomplete lysis, or it is bcos of the inclusion bodies? What if I am not inducing and there is only leaky expression of protein? Is it possible to have inclusion bodies in uninduced sample?
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