I'll second limited proteolysis with chymotrypsin. Best wishes, Jon Cooper. jon.b.coo...@protonmail.com
Sent from Proton Mail Android -------- Original Message -------- On 10/01/2025 07:02, Jeroen Mesters wrote: > Hi, > > three things come to mind besides what has been mentioned already > > - many proteins that have an affinity for nucleic acids actually love > sulfates or phosphates… And yes, phosphates may lead to salt crystals at > higher pH values but this should not stop you a priori from screening with > the same (conditions under-represented in most screens…) > > - what is the pI of your protein? Is it very basic? If so, apply lessons > learned from the Hofmeister series on ions and their effect on protein > stability and solubility. Solubility of basic proteins (pI > 9) can be > improved by adding some ammonium sulfate (100-200 mM in the protein buffers) > and pay special attention to crystal screening solutions/conditions with > thiocyanate, nitrate and chloride as these are good ions that stabilise basic > proteins (and thereby promote crystal formation) > > - what does a sequence comparison shown with homologues that are in the > database? Does the protein by chance have long(er) N and/or C- termini or a > large loop? Limited proteolytic digestion of the protein followed by SDS-PAGE > analysis will be helpful in identifying disruptive factors. > > Good luck, > > Jeroen > > __ > Dr. math. et dis. nat. Jeroen R. Mesters > Biological Safety Officer (BBS) > Deputy, Lecturer, Program Coordinator Infection Biology > Visiting Professorship in Biophysics - University South Bohemia > University of Lübeck > Center for Structural and Cell Biology inMedicine > Institute of Biochemistry > Ratzeburger Allee 160 > 23562 Lübeck > > Tel +49 451 3101 3105 > https://orcid.org/0000-0001-8532-6699 > >> Am 10.01.2025 um 02:00 schrieb Marco Bravo >> <0000d0eb7bee83ae-dmarc-requ...@jiscmail.ac.uk>: >> >> Hello all, >> I am seeking some advice or ideas to crystalize a nuclease protein. It >> exists within a heterodimer, homodimer, and maybe monomer as well depending >> on the species from which the protein is in. The protein has been shown to >> have nuclease activity on its own depending on the species. I am screening >> the protein from three different species and have setup so many crystal >> high-throughput crystal screens with and without DNA. I never get protein >> crystals for these proteins, I have done thermal shift assays to identify a >> buffer that stabilizes the protein better than the storage buffer and found >> some conditions that increased the melting temp by 4-5 degrees. I screened >> the protein again in these new stabilizing buffers but still have not gotten >> any hits. There are protein structures for the protein in the pdb with its >> partner protein in the heterodimer formation. I have experience screening >> other proteins and getting several hits per kit so I know what to expect for >> a successful screen. Any advice would be helpful, I want to retain the >> full-length protein for structural studies. I would appreciate any ideas or >> advice for moving forward and trying to obtain crystals. Thanks! >> >> Marco >> >> ######################################################################## >> >> 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 ######################################################################## 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/
