I realised afterwards that this might be an experimental example of desperately trying to turn a negative result into a positive one, which is something that has been discussed here recently (sorry, Dale) ;-0 ;-0
Sent from ProtonMail mobile -------- Original Message -------- On 24 Apr 2021, 14:33, Jon Cooper wrote: > 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 ######################################################################## 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/
