Some ligands/ small molecules solubility is pH- dependent. (E.g. biotin, EDTA)
Try adjusting the pH (it doesn't have to be accurate, just use a pH
paper if you have very small volumes). It might help you out to
improve ligand solubility (and binding with your protein).
Also, not to ruin the party, but have you done a base line ITC
measurement (protein to buffer, no ligand) and substracted it from the
protein - ligand measurement? It might improve your calculated kd
slightly, or, alas, you'll realise the measured affinity was due to
protein's denaturing upon interaction with the solvent.
Good luck,
Amit
Quoting Jon Cooper <0000488a26d62010-dmarc-requ...@jiscmail.ac.uk>:
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/
########################################################################
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/
