If you want to try co-crystallisation again, if you dissolve your ligand in say DMSO or anything that works, e.g. isopropanol, then add it to your protein up to its maximum tolerable level (i.e. the level up to which the protein is not denatured/inactivated by the solvent - you need to test this), the ligand will start to precipitate out on a grand scale but don't worry, stir the mix at 4°C for a couple of days so that the ligand can slowly partition into the protein binding sites, hopefully. Then you can dialyse out the organic solvent and/or use one of the spin concentrators to remove it and concentrate the protein for crystallisation. There will be tons of precipitated ligand which you can centrifuge out whenever necessary.
Sent from ProtonMail mobile -------- Original Message -------- On 24 Apr 2021, 13:08, abhimanyu singh wrote: > You may soak the crystals with ligands in maximum tolerable DMSO percentage > and harvest crystals at different time points. In my experience working with > some poor affinity, highly hydrophobic compounds, it may take up-to several > days for the ligand to bind with good occupancy (in one instance it was four > days). This may also depend on the crystal soaking/growth temperature. > Ethylene glycol is a good alternative to DMSO in some cases, which might > particularly be helpful in co-crystallisation experiments. > > Greetings, > Abhi > > On Sat, 24 Apr 2021 at 09:11, Casper Wilkens > <00005d34ab0aef35-dmarc-requ...@jiscmail.ac.uk> wrote: > >> How long did you wait before freezing the crystal? Sometimes I have to wait >> days before the ligand finds it way into the binding site. >> >> Casper Wilkens >> Asst. Prof. >> Structural Enzymology & Biorefining >> DTU Bioengineering >> >> Technical University of Denmark >> >> Department of Biotechnology and Biomedicine >> Søltofts Plads >> Building 224 >> Room 028 >> 2800 Kgs. Lyngby >> [c...@dtu.dk](mailto:c...@bio.dtu.dk) >> www.bioengineering.dtu.dk/ >> https://www.cazypedia.org/index.php/User:Casper_Wilkens >> https://twitter.com/protein_artist >> https://www.researchgate.net/profile/Casper_Wilkens >> https://www.cazypedia.org/index.php/User:Casper_Wilkens >> >> --------------------------------------------------------------- >> >> Fra: CCP4 bulletin board <CCP4BB@JISCMAIL.AC.UK> på vegne af Gourab Basu >> Choudhury <goura...@csiriicb.res.in> >> Sendt: 24. april 2021 07:40:20 >> Til: CCP4BB@JISCMAIL.AC.UK >> Emne: Re: [ccp4bb] Co crystalization with less soluble ligand. >> >> I tried sokaing ,high concentration of DMSO is effecting crystal, so I tried >> other contidtion where 10%DMSO is there. Got 1.9 A data with protein co >> crystalization with 10mM ligand. But ligand occupancy is not visble. >> I got the KD value from ITC, with reverse titration. With 10uM ligand in >> cell and 150uM protein in syringe. . >> >> Can anybody suggest some views.please feel free to share your experiences. >> >> On Sat, Apr 24, 2021, 10:49 AM Peat, Tom (Manufacturing, Parkville) >> <tom.p...@csiro.au> wrote: >> >>> Hello Gourab, >>> >>> DMSO is not the only possible solvent- there are others to try. >>> I don't want to sound negative, but 40 micromolar is not a very tight >>> binding compound. It would also depend on how that binding constant was >>> measured- how did someone get enough in solution to measure that? >>> Best of luck, tom >>> >>> Tom Peat, PhD >>> Proteins Group >>> Biomedical Program, CSIRO >>> [343 Royal >>> Parade](https://www.google.com/maps/search/343+Royal+Parade+%0D%0AParkville,+VIC,+3052?entry=gmail&source=g) >>> [Parkville, VIC, >>> 3052](https://www.google.com/maps/search/343+Royal+Parade+%0D%0AParkville,+VIC,+3052?entry=gmail&source=g) >>> +613 9662 7304 >>> +614 57 539 419 >>> tom.p...@csiro.au >>> >>> --------------------------------------------------------------- >>> >>> From: CCP4 bulletin board <CCP4BB@JISCMAIL.AC.UK> on behalf of Gourab Basu >>> Choudhury <goura...@csiriicb.res.in> >>> Sent: Saturday, April 24, 2021 12:46 PM >>> To: CCP4BB@JISCMAIL.AC.UK <CCP4BB@JISCMAIL.AC.UK> >>> Subject: [ccp4bb] Co crystalization with less soluble ligand. >>> >>> Hello everyone, >>> >>> I am finding it difficult for getting a ligand density inside the protein >>> as the ligand is very much insoluble. It's only soluble in 100% DMSO. I >>> tried for co crystalization. Kd value of the ligand is near 40um. Any >>> suggestion what to do? >>> >>> --------------------------------------------------------------- >>> >>> To unsubscribe from the CCP4BB list, click the following link: >>> https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1 >> >> --------------------------------------------------------------- >> >> To unsubscribe from the CCP4BB list, click the following link: >> https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1 >> >> --------------------------------------------------------------- >> >> To unsubscribe from the CCP4BB list, click the following link: >> https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1 > > -- > > Abhimanyu Kumar Singh, PhD > Post-doctoral Research Fellow > Laboratory of Virology and Chemotherapy, Rega Institute for Medical Research > Department of Microbiology and Immunology, KU Leuven > Herestraat 49, box 1030, 3000 Leuven > Belgium. > > --------------------------------------------------------------- > > To unsubscribe from the CCP4BB list, click the following link: > https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1 ######################################################################## 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/
