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.

[log in to unmask]">

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]>:

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]

--------------------------------------------