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 <https://www.douglas.co.uk/PDB_data.htm> Good luck, Patrick [image: image.png] On Mon, Feb 5, 2024 at 10:27 AM kavyashreem <[email protected]> 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 > -- [email protected] 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. GB 480 7371 36 ######################################################################## To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1 This message was issued to members of www.jiscmail.ac.uk/CCP4BB, a mailing list hosted by www.jiscmail.ac.uk, terms & conditions are available at https://www.jiscmail.ac.uk/policyandsecurity/
