[ccp4bb] Setting drops with proteins that are hard to concentrate
Dear All, I have a 50kDa protein that is soluble and monodisperse at up to approx 1mg/ml (after Ni-affinity and size-exclusion chromatography). However, it aggregates (probably both via disulphides and via 'sticky/hydrophobic patches') when I concentrate it towards 2-3mg/ml, even in the presence of several detergents. I don't want to add DTT since my protein should have several intramolecular disulphidesalthough I do have 2 free Cysteines, partially exposed. I have already tried mutating the Cysteines, with little improvement. Any suggestions for obtaining 5-10mg/ml? Does anybody have good experiences with usin L-Arg and L-Glu (e.g. At 50mM) to aid concentrating (as in the Golovanov AP paper, JACS, 2004, pages 8933...) Thanks for any input! Yours, Matt Matthew J. Bottomley, Ph.D. Senior Research Biochemist IRBM / MRL-Rome Notice: This e-mail message, together with any attachments, contains information of Merck Co., Inc. (One Merck Drive, Whitehouse Station, New Jersey, USA 08889), and/or its affiliates (which may be known outside the United States as Merck Frosst, Merck Sharp Dohme or MSD and in Japan, as Banyu - direct contact information for affiliates is available at http://www.merck.com/contact/contacts.html) that may be confidential, proprietary copyrighted and/or legally privileged. It is intended solely for the use of the individual or entity named on this message. If you are not the intended recipient, and have received this message in error, please notify us immediately by reply e-mail and then delete it from your system.
Re: [ccp4bb] Setting drops with proteins that are hard to concentrate
I have a similar problem Matthew. Just got some NVoy from Novexin in which some claim can help if hydrophobic patches are the main problem. Will see. Regards, Roberto On 24 Apr 2008, at 12:35, Bottomley, Matthew wrote: Dear All, I have a 50kDa protein that is soluble and monodisperse at up to approx 1mg/ml (after Ni-affinity and size-exclusion chromatography). However, it aggregates (probably both via disulphides and via 'sticky/hydrophobic patches') when I concentrate it towards 2-3mg/ ml, even in the presence of several detergents. I don't want to add DTT since my protein should have several intramolecular disulphidesalthough I do have 2 free Cysteines, partially exposed. I have already tried mutating the Cysteines, with little improvement. Any suggestions for obtaining 5-10mg/ml? Does anybody have good experiences with usin L-Arg and L-Glu (e.g. At 50mM) to aid concentrating (as in the Golovanov AP paper, JACS, 2004, pages 8933...) Thanks for any input! Yours, Matt Matthew J. Bottomley, Ph.D. Senior Research Biochemist IRBM / MRL-Rome Notice: This e-mail message, together with any attachments, contains information of Merck Co., Inc. (One Merck Drive, Whitehouse Station, New Jersey, USA 08889), and/or its affiliates (which may be known outside the United States as Merck Frosst, Merck Sharp Dohme or MSD and in Japan, as Banyu - direct contact information for affiliates is available at http://www.merck.com/ contact/contacts.html) that may be confidential, proprietary copyrighted and/or legally privileged. It is intended solely for the use of the individual or entity named on this message. If you are not the intended recipient, and have received this message in error, please notify us immediately by reply e-mail and then delete it from your system. --- Dr. Roberto Steiner Randall Division of Cell and Molecular Biophysics New Hunt's House King's College London Guy's Campus London, SE1 1UL Phone +44 (0)20-7848-8216 Fax +44 (0)20-7848-6435 e-mail [EMAIL PROTECTED]
Re: [ccp4bb] Setting drops with proteins that are hard to concentrate
can't you try different volumes: i.e. 360 nl protein solution in very little buffer and salt + 40 nl precipitant. Should give roughly 10 mg/ml after equilibration. Best wishes Kornelius On Thu, 24 Apr 2008 12:42:42 +0100 Roberto Steiner [EMAIL PROTECTED] wrote: I have a similar problem Matthew. Just got some NVoy from Novexin in which some claim can help if hydrophobic patches are the main problem. Will see. Regards, Roberto On 24 Apr 2008, at 12:35, Bottomley, Matthew wrote: Dear All, I have a 50kDa protein that is soluble and monodisperse at up to approx 1mg/ml (after Ni-affinity and size-exclusion chromatography). However, it aggregates (probably both via disulphides and via 'sticky/hydrophobic patches') when I concentrate it towards 2-3mg/ ml, even in the presence of several detergents. I don't want to add DTT since my protein should have several intramolecular disulphidesalthough I do have 2 free Cysteines, partially exposed. I have already tried mutating the Cysteines, with little improvement. Any suggestions for obtaining 5-10mg/ml? Does anybody have good experiences with usin L-Arg and L-Glu (e.g. At 50mM) to aid concentrating (as in the Golovanov AP paper, JACS, 2004, pages 8933...) Thanks for any input! Yours, Matt Matthew J. Bottomley, Ph.D. Senior Research Biochemist IRBM / MRL-Rome Notice: This e-mail message, together with any attachments, contains information of Merck Co., Inc. (One Merck Drive, Whitehouse Station, New Jersey, USA 08889), and/or its affiliates (which may be known outside the United States as Merck Frosst, Merck Sharp Dohme or MSD and in Japan, as Banyu - direct contact information for affiliates is available at http://www.merck.com/ contact/contacts.html) that may be confidential, proprietary copyrighted and/or legally privileged. It is intended solely for the use of the individual or entity named on this message. If you are not the intended recipient, and have received this message in error, please notify us immediately by reply e-mail and then delete it from your system. --- Dr. Roberto Steiner Randall Division of Cell and Molecular Biophysics New Hunt's House King's College London Guy's Campus London, SE1 1UL Phone +44 (0)20-7848-8216 Fax +44 (0)20-7848-6435 e-mail [EMAIL PROTECTED] -- Kornelius Zeth Max Planck Institute for Developmental Biology Dept. Protein Evolution Spemannstr. 35 72076 Tuebingen, Germany [EMAIL PROTECTED] Tel -49 7071 601 323 Fax -49 7071 601 349
Re: [ccp4bb] Setting drops with proteins that are hard to concentrate
Dear Matt, make sure that you don't have slow or even fast formation of unspecific intermolecular disulfides by simple checks at several time points on SDS-PAGE using sample buffer without DTT. Check the content of free thiols and disulfides of your protein over a period of time. If you need easy and solid protocols, just send me a mail. Guenter Bottomley, Matthew wrote: Dear All, I have a 50kDa protein that is soluble and monodisperse at up to approx 1mg/ml (after Ni-affinity and size-exclusion chromatography). However, it aggregates (probably both via disulphides and via 'sticky/hydrophobic patches') when I concentrate it towards 2-3mg/ml, even in the presence of several detergents. I don't want to add DTT since my protein should have several intramolecular disulphidesalthough I do have 2 free Cysteines, partially exposed. I have already tried mutating the Cysteines, with little improvement. Any suggestions for obtaining 5-10mg/ml? Does anybody have good experiences with usin L-Arg and L-Glu (e.g. At 50mM) to aid concentrating (as in the Golovanov AP paper, JACS, 2004, pages 8933...) Thanks for any input! Yours, Matt Matthew J. Bottomley, Ph.D. Senior Research Biochemist IRBM / MRL-Rome Notice: This e-mail message, together with any attachments, contains information of Merck Co., Inc. (One Merck Drive, Whitehouse Station, New Jersey, USA 08889), and/or its affiliates (which may be known outside the United States as Merck Frosst, Merck Sharp Dohme or MSD and in Japan, as Banyu - direct contact information for affiliates is available at http://www.merck.com/contact/contacts.html) that may be confidential, proprietary copyrighted and/or legally privileged. It is intended solely for the use of the individual or entity named on this message. If you are not the intended recipient, and have received this message in error, please notify us immediately by reply e-mail and then delete it from your system. -- *** Priv.Doz.Dr. Guenter Fritz Fachbereich Biologie Sektion Naturwissenschaften Universitaet Konstanz http://www.biologie.uni-konstanz.de/fritz Universitaetsstrasse 10 Postfach M665 D-78457 Konstanz e-mail: [EMAIL PROTECTED] Tel. Office: +49-(0)7531 88 3205 Tel. Lab : +49-(0)7531 88 3687 Fax: +49-(0)7531 88 2966
Re: [ccp4bb] Setting drops with proteins that are hard to concentrate
Dear Matt, In one of our project, the protein of interest used to elute from Ni-column with ~20 other proteins. I also guessed it due to surface Cysteines. Addition of 2mM free Cys in the protein sample helped, which competed with free Cys on the protein surface and, ultimately, gave happy single band on sds-page. All the best Manish -- Center for Advanced Research in Biotechnology University of Maryland Biotechnology Institute 9600 Gudelsky Drive, Rockville MD 20850 USA Tel: +1-240-314-6130; fax: +1-240-314-6255 - Original Message From: Bottomley, Matthew [EMAIL PROTECTED] To: CCP4BB@JISCMAIL.AC.UK Sent: Thursday, April 24, 2008 7:35:46 AM Subject: [ccp4bb] Setting drops with proteins that are hard to concentrate Setting drops with proteins that are hard to concentrate Dear All, I have a 50kDa protein that is soluble and monodisperse at up to approx 1mg/ml (after Ni-affinity and size-exclusion chromatography). However, it aggregates (probably both via disulphides and via 'sticky/hydrophobic patches') when I concentrate it towards 2-3mg/ml, even in the presence of several detergents. I don't want to add DTT since my protein should have several intramolecular disulphidesalthough I do have 2 free Cysteines, partially exposed. I have already tried mutating the Cysteines, with little improvement. Any suggestions for obtaining 5-10mg/ml? Does anybody have good experiences with usin L-Arg and L-Glu (e.g. At 50mM) to aid concentrating (as in the Golovanov AP paper, JACS, 2004, pages 8933...) Thanks for any input! Yours, Matt Matthew J. Bottomley, Ph.D. Senior Research Biochemist IRBM / MRL-Rome Be a better friend, newshound, and know-it-all with Yahoo! Mobile. Try it now. http://mobile.yahoo.com/;_ylt=Ahu06i62sR8HDtDypao8Wcj9tAcJ
