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Dear Eugene, the additive is a metal-polyanion with a net charge of 6- ... yes, NaCl affects largely the solubility of the anions, however, the LLPS appears within a rather large protein concentration range (10-50 mg/ml = 0.7 - 3.5 mM) in the presence of e.g. 0.1, 0.5, 1.0, 2.5 and 5.0 mM additive but only at NaCl conc = 0.25 M. I will set up conditions with higher additive conc to check if the LLPS-region changes with the amount of additive. Am 09.12.2018 um 14:23 schrieb Eugene Osipov: > Hi, Alex, > you did not mention exactly what kind of addictive you use. I suggest > that amorphous precipitation is due to this addictive as proteins more > likely to precipitate in presence of polyvalent ions. > May I suggest that sodium chloride could affect solubility of you > anions and thus zone with higher protein solubility-lower addictive > contentration are responsible for observed LLPS > > сб, 8 дек. 2018 г. в 14:52, Aleksandar Bijelic > <[log in to unmask] > <mailto:[log in to unmask]>>: > > Dear CCP4 Community, > > First of all, I want to aplogize in advance for this more or less > off-topic request. I am currently investigating the phase behavior of > Lysozyme (HEWL) in the presence of NaCl and an anionic metal cluster > (additive) using the microbatch under oil technique. Before the > experiment I expected that the additive will might lead to a shift of > the phase boundaries in comaprison to the HEWL-NaCl system, or > maybe to > an increase of the phase space, where nucleation or even crystals > occur. > Unfortunately, the HEWL-NaCl-cluster-system did not exhibit a > textbook-example of a phase diagram as at almost every condition > (different protein, salt and cluster conc.) an amorphous > precipitation > was immediately formed, which in most of the cases became crystalline > within 1-5 days (mostly shower of needles, spherulites and sea > urchins > and sometimes crystals). The transformation from amorphous to > crystalline precipitate was accompanied by liquid-liquid-phase > separation (LLPS), i.e. the amorphous precipitates dissovled > within 1-2 > days and LLPS was observed before the crystalline precipitate was > formed. The odd thing is that LLPS was always observed at the same > NaCl > concentration (0.25-0.35 M, but mostly 0.25 M) independent of the > protein or cluster concentration. At the beginning I thought that > I was > located at the edge of a very narrow LLPS-region, however, testing at > higher protein conc. did not change or shift the LLPS conditions > as in > the range of 10-50 mg/ml HEWL (0-50 mg/ml was investigated) and > independet of the cluster conc. (0.1 - 5.0 mM), the LLPS occured > always > at 0.25-0.35 M NaCl. As I am far away from being an expert in protein > phase behavior, I cannot explain this "magical" salt conc. that > induces > at every tested protein and cluster conc. LLPS. Thus, I hope that > somebody of you might have observed the same or a similar behavior > and > is able to explain this to me. Thanks in advance! > > Regards, > > Aleks > > ######################################################################## > > To unsubscribe from the CCP4BB list, click the following link: > https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB&A=1 > > > > -- > Eugene Osipov > Junior Research Scientist > Laboratory of Enzyme Engineering > Research Center of Biotechnology -- ------------------------------------------- Dr. Aleksandar Bijelic Institut für Biophysikalische Chemie Universität Wien Althanstrasse 14 A-1090 Wien Tel: +43 1 4277 52533 e-Mail:[log in to unmask] -------------------------------------------- ######################################################################## To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB&A=1