Dear Patrick, Thank you for the suggestions and for the detailed the statistics!!
Regards Kavya On 2024-02-06 23:06, Patrick Shaw Stewart wrote: > Hi Kavya > > 1. Make a seed stock from the globules or anything else that you think might > be crystalline, and recreen. In other words, you should add your seed stock > to _random screens_ (not optimization experiments). There could be many > conditions that are in the metastable zone of the phase diagram in your > normal screens - this method can give you crystals in those conditions. > > If this works, you'll be in a better position anyway because you'll have more > control - by diluting the seed stock, you can control the number of crystals > per drop. > > References: > >> D'Arcy, A., Villard, F. and Marsh, M., 2007. An automated microseed >> matrix-screening method for protein crystallization. _Acta Crystallographica >> Section D: Biological Crystallography_, _63_(4), pp.550-554. >> >> Shaw Stewart, P.D., Kolek, S.A., Briggs, R.A., Chayen, N.E. and Baldock, >> P.F., 2011. Random microseeding: a theoretical and practical exploration of >> seed stability and seeding techniques for successful protein >> crystallization. _Crystal Growth & Design_, _11_(8), pp.3432-3441. > > This is how we normally make the seed: > >> https://www.douglas.co.uk/f_ftp1/rMMS_Procedure.pdf > > 2. Many years ago, we did some data mining of the crystallization conditions > in a remark in the PDB. The concentrations that people reported are below. > There were eight reports where over 100 mg/mL was used. It only goes up to > 2004. > >> https://www.douglas.co.uk/PDB_data.htm [1] > > Good luck, > > Patrick > > On Mon, Feb 5, 2024 at 10:27 AM kavyashreem <kavyashr...@instem.res.in> > wrote: > >> Dear All, >> >> Has anyone worked on a protein which is highly soluble even at 80mg/ml? >> >> We have one such candidate, which does not precipitate even at 80mg/ml >> instead forms phase separated globules in crystallization plate, which >> eventually hardens over a period of 1 to 1.5 months (which is florescent >> under UV microscope.) >> >> We tried screening at different pH, but failed to get any hits. >> >> Since we got few conditions in which the phase separated globules >> solidified, we focused on them and expanded with 120mg/ml protein, still >> there were not visible precipitates except for the phase separation. This >> has been a challenging target so far. We have tried with different >> constructs, which unfortunately are not soluble! >> >> Does POMs help in such cases? Or do you have any other suggestion. >> >> Thank you >> >> Regards >> >> Kavya >> >> CONFIDENTIAL: THIS EMAIL AND ANY FILES TRANSMITTED WITH IT ARE CONFIDENTIAL >> AND INTENDED SOLELY FOR THE USE OF THE INDIVIDUAL OR GROUP TO WHOM THEY ARE >> ADDRESSED. IF YOU HAVE RECEIVED THIS EMAIL IN ERROR, PLEASE NOTIFY THE >> SENDER IMMEDIATELY AND DELETE THIS E-MAIL FROM YOUR SYSTEM. IT IS NOT >> AUTHORISED TO COPY, FORWARD, OR IN ANY WAY REVEAL THE CONTENTS OF THIS >> MESSAGE TO ANYONE. >> >> ------------------------- >> >> To unsubscribe from the CCP4BB list, click the following link: >> https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1 > > -- > > patr...@douglas.co.uk Douglas Instruments Ltd. > Douglas House, East Garston, Hungerford, Berkshire, RG17 7HD, UK > Directors: Patrick Shaw Stewart, Peter Baldock, Stefan Kolek > > http://www.douglas.co.uk > Tel: 44 (0) 148-864-9090 US toll-free 1-877-225-2034 > Regd. England 2177994, VAT Reg. 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