Re: [ccp4bb] Off Topic- Cystine Detection
Am Mittwoch 06 Februar 2013 17:10:35 schrieb Yuri Pompeu: Dear All, I am trying to probe the existence of a disulfide bond on the surface of my protein. I have attempted Ellman´s and my results were not as clear as I would have hoped for. I am not a sulfur/cysteine chemist and would appreciate the advice on what experiments to try! Thanks a bunch YAP Hi, You might also try ABDF labeling of free cysteines. Separate the protein on a native gel and look which of your bands show fluorescence under UV. If you compare it to a protein with known free cysteines, you might also get an idea how much closed your disulfide bridge is actually. Christian
Re: [ccp4bb] Off Topic- Cystine Detection
Perhaps you should have a look at http://193.146.160.29/gtb/sod/usu/$UBUG/repositorio/10320076_Hawkins.pdf. and at An introduction to methods for analyzing thiols and disulfides: Reactions, reagents, and practical considerations. Rosa E. Hansen 1, Jakob R. Winther Analytical Biochemistry 394 (2009) 147–158. HTH, Nadir Mrabet Pr. Nadir T. Mrabet Structural Molecular Biochemistry N-gere - INSERM U-954 University of Lorraine, Nancy School of Sciences and Technologies School of Medicine 9, Avenue de la Foret de Haye, BP 184 54505 Vandoeuvre-les-Nancy Cedex France Phone: +33 (0)3.83.68.32.73 Fax: +33 (0)3.83.68.32.79 E-mail: Nadir.Mrabet at univ-lorraine.fr On 07/02/2013 04:17, Dr. Anthony Addlagatta wrote: *** This message has been scanned by the InterScan for CSC SSM by IICT security policy and found to be free of known security risks. *** Dear Yuri, If you have access to mass spec, this should be a straight forward experiment. Find the reference here. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2291582/pdf/v010p00017.pdf What was the result in the Ellman´s reaction? Unless you have other reactive cysteines in your protein protein, you should not see any color if the pair of cysteines on surface form disulfide. Anthony - Dr. Anthony Addlagatta Center for Chemical Biology Indian Institute of Chemical Technology [IICT] Tarnaka, Hyderabad AP-500 607, INDIA Tel:91-40-27191812 Web: https://sites.google.com/site/chembioliict/home/dr-anthony-addlagatta-1 -- Original Message --- From: Yuri Pompeu yuri.pom...@ufl.edu To: CCP4BB@JISCMAIL.AC.UK Sent: Wed, 6 Feb 2013 16:10:35 + Subject: [ccp4bb] Off Topic- Cystine Detection *** This message has been scanned by the InterScan for CSC SSM at IICT and found to be free of known security risks. *** Dear All, I am trying to probe the existence of a disulfide bond on the surface of my protein. I have attempted Ellman´s and my results were not as clear as I would have hoped for. I am not a sulfur/cysteine chemist and would appreciate the advice on what experiments to try! Thanks a bunch YAP --- End of Original Message --- This Mail Scanned by ClamAV and Spammassassin
Re: [ccp4bb] Off Topic- Cystine Detection
Even so, it should run somewhat differently, and probably visibly on SDS-PAGE. Even a single phosphorylation changes mobility, and I think I remember having seen intramolecular disulfides change mobility (anyone else have experience on this?) JPK On Wed, Feb 6, 2013 at 10:33 PM, Yuri yuri.pom...@ufl.edu wrote: ** The disulfide bond is intramolecular. I do not have reasons to believe it is cross linking my protein On Wed, 6 Feb 2013 22:16:36 -0500, Jacob Keller wrote: Couldn't you just run reducing/non-reducing SDS-PAGE lanes and see the difference? JPK On Wed, Feb 6, 2013 at 11:10 AM, Yuri Pompeu yuri.pom...@ufl.edu wrote: Dear All, I am trying to probe the existence of a disulfide bond on the surface of my protein. I have attempted Ellman´s and my results were not as clear as I would have hoped for. I am not a sulfur/cysteine chemist and would appreciate the advice on what experiments to try! Thanks a bunch YAP -- *** Jacob Pearson Keller, PhD Postdoctoral Associate HHMI Janelia Farms Research Campus email: j-kell...@northwestern.edu *** -- Yuri Pompeu -- *** Jacob Pearson Keller, PhD Postdoctoral Associate HHMI Janelia Farms Research Campus email: j-kell...@northwestern.edu ***
Re: [ccp4bb] Off Topic- Cystine Detection
I never done such experiments but N-Ethylmaleimide labeling and Mass-Spec could be the solution. Le 6 févr. 2013 à 17:10, Yuri Pompeu yuri.pom...@ufl.edu a écrit : Dear All, I am trying to probe the existence of a disulfide bond on the surface of my protein. I have attempted Ellman´s and my results were not as clear as I would have hoped for. I am not a sulfur/cysteine chemist and would appreciate the advice on what experiments to try! Thanks a bunch YAP Xavier Brazzolotto, PhD Département de Toxicologie Institut de Recherche Biomédicale des Armées 24 Av des Maquis du Grésivaudan 38702 La Tronche Cedex France
Re: [ccp4bb] Off Topic- Cystine Detection
Hi, to my knowledge, Ellman's reagent detects free thiols only. (Or am I wrong?) In contrast, with the method of Thannhauser (Thannhauser [1987], Methods Enzymol. 143, 115-9) you can determine the total amount of thiols (free and in disulfide bonds). Any difference between the two methods should indicate the presence of disulfide bonds, but will probably not allow for their actual quantification. Regards, Joern ** Address: Joern Krausze Molecular Structural Biology Helmholtz Centre for Infection Research Inhoffenstrasse 7 38124 Braunschweig Germany Email: joern.krau...@helmholtz-hzi.de Phone: +49 (0)531 6181 7023 (office) +49 (0)531 6181 7020 (lab) ** On Wed, 6 Feb 2013, Yuri Pompeu wrote: Dear All, I am trying to probe the existence of a disulfide bond on the surface of my protein. I have attempted Ellman´s and my results were not as clear as I would have hoped for. I am not a sulfur/cysteine chemist and would appreciate the advice on what experiments to try! Thanks a bunch YAP
Re: [ccp4bb] Off Topic- Cystine Detection
Couldn't you just run reducing/non-reducing SDS-PAGE lanes and see the difference? JPK On Wed, Feb 6, 2013 at 11:10 AM, Yuri Pompeu yuri.pom...@ufl.edu wrote: Dear All, I am trying to probe the existence of a disulfide bond on the surface of my protein. I have attempted Ellman´s and my results were not as clear as I would have hoped for. I am not a sulfur/cysteine chemist and would appreciate the advice on what experiments to try! Thanks a bunch YAP -- *** Jacob Pearson Keller, PhD Postdoctoral Associate HHMI Janelia Farms Research Campus email: j-kell...@northwestern.edu ***
Re: [ccp4bb] Off Topic- Cystine Detection
*** This message has been scanned by the InterScan for CSC SSM by IICT security policy and found to be free of known security risks. *** Dear Yuri, If you have access to mass spec, this should be a straight forward experiment. Find the reference here. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2291582/pdf/v010p00017.pdf What was the result in the Ellman´s reaction? Unless you have other reactive cysteines in your protein protein, you should not see any color if the pair of cysteines on surface form disulfide. Anthony - Dr. Anthony Addlagatta Center for Chemical Biology Indian Institute of Chemical Technology [IICT] Tarnaka, Hyderabad AP-500 607, INDIA Tel:91-40-27191812 Web: https://sites.google.com/site/chembioliict/home/dr-anthony-addlagatta-1 -- Original Message --- From: Yuri Pompeu yuri.pom...@ufl.edu To: CCP4BB@JISCMAIL.AC.UK Sent: Wed, 6 Feb 2013 16:10:35 + Subject: [ccp4bb] Off Topic- Cystine Detection *** This message has been scanned by the InterScan for CSC SSM at IICT and found to be free of known security risks. *** Dear All, I am trying to probe the existence of a disulfide bond on the surface of my protein. I have attempted Ellman´s and my results were not as clear as I would have hoped for. I am not a sulfur/cysteine chemist and would appreciate the advice on what experiments to try! Thanks a bunch YAP --- End of Original Message --- This Mail Scanned by ClamAV and Spammassassin
