I am purifying c-Myc partial protein after overexpression in Rosetta cells. 8 M urea precipitates at lower temperatures so I attempted to lyse the Rosetta cells overnight at room temperature by gentle rotation, hoping that the protein would remain stable. Protocol has mentioned lysis for 15-30 mins at room temperature, but I have experienced that this period is not enough for cell lysis.
I would really appreciate any suggestion regarding the proper method for lysis using 8M urea.
Perhaps, you can use 6M guanidine hydrochloride instead of 8M urea. A better chaotrope than urea an d stable at low temperature.
But I do prefer lysis bacterial cells by freeze/thaw cycles and sonication. Then fuge , check the supernatant and resuspend the pellet in a chaotrope buffer ( guanidine or urea ).
Hi there,
Urea on its own is not enough to efficiently disrupt cells and is rather used to denaturate and solubilize proteins . You should combine it with either mechanical lysis(sonication, thaw/freezing) or enzymatic/chemical lysis(lysozyme+detergent treatment, alkaline lysis)...
4-6M guanidine hydrochloride lysis buffer with ultrasonic bath, or thawing/freezing, or even vortexing is not the cheapest but perfect for any protein extraction method. Overnight room temperature lysis is not a good idea anyway, more of all proteins will be disrupted than extracted without guanidine. Having urea only you should either homogenize cell membranes with sonication or freezing-thawing, or digest them with SDS-lysozyme buffer or similar - but quickly.
There a nice protocols in the NMR papers of Konrat to purify some myc constructs.Initial lysis in a non denaturing buffer to wash the inclusion bodies.Then you probably just need 8 M Urea and the other ingredients which are in the protocol.I personally purified myc fragments via coexpression with max, if they contain the heterodimerization motif.Then it is soluble and you get it very pure first.Then get rid of max via denaturation and another column.Described in his JMB paper 2001.
Thank you for your suggestions. I would try to follow and then check how it works.
Actually I use 8M urea combined with sodium phosphate monobasic plus Tris base to make a lysis buffer as mentioned in the QIAGEN, puriciation of 6X His tagged proteins, manual.
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