Hi Prerana, You remind me of my struggle to obtain the crystal of mushroom tyrosinase years ago. Exactly the same situation, I always got precipitation in almost every condition (some were just clear drops, which stayed clear forever until they dried out). In despair (after years of unsuccessful attempts), I carelessly purified the enzyme in water and used the purified enzyme for crystallization. Amazingly, I found crystals in some conditions where the crystal had never grew before. The crystals diffracted badly (about 8 Angstrom) but this initial finding led us to purify the enzyme using buffer with low ionic strength and set up crystallization: Optimization of purification conditions (with thermofluor) brought us to the use of HEPES buffer, which indeed has a very low ionic strength. In addition, the strikingly strange result of thermofluor assay was, under the original buffer condition the enzyme has a nice melting temperature profile (the presence of calcium and HEPES made it even better, shift the Tm to a higher temperature) but the enzyme in water displayed a profile of unstructured protein.
Wangsa et. al. (2011) Acta Crystallogr. F 67, 575-578. Good luck. Wangsa On Fri, Feb 21, 2014 at 6:14 PM, Prerana G. <[email protected]> wrote: > Hi, Sorry for asking an off-topic question, > I have recently purified a protein having a molecular weight of 40kDa and > concentration of the protein was 8mg/ml. When I tried to set the protein > for crystallisation using micobatch method, the protein started > precipitating in most of the buffer conditions of Crystal screen and Peg > ion. The precipitation took place very quickly (within 5-10 mins). > How should I overcome this problem? > > > Regards > Prerana > -- ================ Wangsa Tirta Ismaya Jakarta, Indonesia
