[ccp4bb] Detergent and protein oligomerization
Dear Raji the detergent micelle increases the hydrodynamic radius of a membrane protein and therefore affects the behavior on SEC. The following papers show examples how the molecular mass and therefore oligomeric state of membrane proteins can be determined by SEC: Slotboom, D. J., Duurkens, R. H., Olieman, K., and Erkens, G. B. (2008). Static light scattering to characterize membrane proteins in detergent solution. Methods 46, 73–82. Kunji, E. R. S., Harding, M., Butler, P. J. G., and Akamine, P. (2008). Determination of the molecular mass and dimensions of membrane proteins by size exclusion chromatography. Methods 46, 62–72. Regards Florian Date:Thu, 21 Jun 2012 11:50:09 -0400 From:Raji Edayathumangalam r...@brandeis.edu Subject: Detergent and protein oligomerization Hi Everyone, Sorry for the non-CCP4 post. I have a very basic question about detergents, critical micelle concentration and behavior on gel filtration. A 33kDa membrane protein was purified by gel filtration in a buffer containing 0.4%(w/v) beta-NG (CMC: 6.5mM) and 0.046%(w/v)LDAO (CMC: 0.14mM). So the concentrations of beta-NG and LDAO in the gel-filtration buffer are ~2X and ~14X that of the CMCs of the respective detergents. The elution volume of the protein peak (plus detergent) on Superdex200 corresponds to a molecular mass of 100kDa. I think that the 100kDa mass above includes contributions from both the protein as well as the detergent micelles. If this is correct, is it then accurate to try to glean the oligomerization state of the protein (and conclude that it is a trimer or tetramer) without taking into account detergent micellar mass and its influence on elution volume? How should one interpret the 100kDa mass estimate from the gel filtration? Thanks. Raji -- Raji Edayathumangalam Instructor in Neurology, Harvard Medical School Research Associate, Brigham and Women's Hospital Visiting Research Scholar, Brandeis University - Dr. Florian Brückner Biomolecular Research Laboratory OFLG/102 Paul Scherrer Institut CH-5232 Villigen PSI Switzerland Tel.: +41-(0)56-310-2332 Email: florian.brueck...@psi.ch
[ccp4bb] Detergent and protein oligomerization
Hi Everyone, Sorry for the non-CCP4 post. I have a very basic question about detergents, critical micelle concentration and behavior on gel filtration. A 33kDa membrane protein was purified by gel filtration in a buffer containing 0.4%(w/v) beta-NG (CMC: 6.5mM) and 0.046%(w/v)LDAO (CMC: 0.14mM). So the concentrations of beta-NG and LDAO in the gel-filtration buffer are ~2X and ~14X that of the CMCs of the respective detergents. The elution volume of the protein peak (plus detergent) on Superdex200 corresponds to a molecular mass of 100kDa. I think that the 100kDa mass above includes contributions from both the protein as well as the detergent micelles. If this is correct, is it then accurate to try to glean the oligomerization state of the protein (and conclude that it is a trimer or tetramer) without taking into account detergent micellar mass and its influence on elution volume? How should one interpret the 100kDa mass estimate from the gel filtration? Thanks. Raji -- Raji Edayathumangalam Instructor in Neurology, Harvard Medical School Research Associate, Brigham and Women's Hospital Visiting Research Scholar, Brandeis University
Re: [ccp4bb] Detergent and protein oligomerization
First of all, isn't the choice either dimer or trimer, and second, as a protein-detergent complex (PDC), it would be very unlikely that a trimer of 99 kD would run at 100 kD, although all is fair in love, war, and membrane proteins. JPK On Thu, Jun 21, 2012 at 10:50 AM, Raji Edayathumangalam r...@brandeis.eduwrote: Hi Everyone, Sorry for the non-CCP4 post. I have a very basic question about detergents, critical micelle concentration and behavior on gel filtration. A 33kDa membrane protein was purified by gel filtration in a buffer containing 0.4%(w/v) beta-NG (CMC: 6.5mM) and 0.046%(w/v)LDAO (CMC: 0.14mM). So the concentrations of beta-NG and LDAO in the gel-filtration buffer are ~2X and ~14X that of the CMCs of the respective detergents. The elution volume of the protein peak (plus detergent) on Superdex200 corresponds to a molecular mass of 100kDa. I think that the 100kDa mass above includes contributions from both the protein as well as the detergent micelles. If this is correct, is it then accurate to try to glean the oligomerization state of the protein (and conclude that it is a trimer or tetramer) without taking into account detergent micellar mass and its influence on elution volume? How should one interpret the 100kDa mass estimate from the gel filtration? Thanks. Raji -- Raji Edayathumangalam Instructor in Neurology, Harvard Medical School Research Associate, Brigham and Women's Hospital Visiting Research Scholar, Brandeis University -- *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program email: j-kell...@northwestern.edu ***
Re: [ccp4bb] Detergent and protein oligomerization
Dear Raji, The best way to find out is to run a SEC-MALLS (Size Exclusion Chomatography - Multi-Angle Laser Light Scattering) experiment. Best, Isabel - Dr. Isabel De Moraes, MRSC Membrane Protein Laboratory Facility Co-ordinator Membrane Protein Laboratory Diamond Light Source Ltd, Chilton, Didcot, Oxfordshire, OX11 ODE, UK Tel (direct): 01235 778664 -- From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Jacob Keller [j-kell...@fsm.northwestern.edu] Sent: 21 June 2012 17:11 To: ccp4bb Subject: Re: [ccp4bb] Detergent and protein oligomerization First of all, isn't the choice either dimer or trimer, and second, as a protein-detergent complex (PDC), it would be very unlikely that a trimer of 99 kD would run at 100 kD, although all is fair in love, war, and membrane proteins. JPK On Thu, Jun 21, 2012 at 10:50 AM, Raji Edayathumangalam r...@brandeis.edumailto:r...@brandeis.edu wrote: Hi Everyone, Sorry for the non-CCP4 post. I have a very basic question about detergents, critical micelle concentration and behavior on gel filtration. A 33kDa membrane protein was purified by gel filtration in a buffer containing 0.4%(w/v) beta-NG (CMC: 6.5mM) and 0.046%(w/v)LDAO (CMC: 0.14mM). So the concentrations of beta-NG and LDAO in the gel-filtration buffer are ~2X and ~14X that of the CMCs of the respective detergents. The elution volume of the protein peak (plus detergent) on Superdex200 corresponds to a molecular mass of 100kDa. I think that the 100kDa mass above includes contributions from both the protein as well as the detergent micelles. If this is correct, is it then accurate to try to glean the oligomerization state of the protein (and conclude that it is a trimer or tetramer) without taking into account detergent micellar mass and its influence on elution volume? How should one interpret the 100kDa mass estimate from the gel filtration? Thanks. Raji -- Raji Edayathumangalam Instructor in Neurology, Harvard Medical School Research Associate, Brigham and Women's Hospital Visiting Research Scholar, Brandeis University -- *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program email: j-kell...@northwestern.edumailto:j-kell...@northwestern.edu ***
Re: [ccp4bb] Detergent and protein oligomerization
There is something even better than SEC-MALS--solving the structure! JPK On Thu, Jun 21, 2012 at 11:16 AM, isabel.de-mor...@diamond.ac.uk wrote: Dear Raji, The best way to find out is to run a SEC-MALLS (Size Exclusion Chomatography - Multi-Angle Laser Light Scattering) experiment. Best, Isabel - Dr. Isabel De Moraes, MRSC Membrane Protein Laboratory Facility Co-ordinator Membrane Protein Laboratory Diamond Light Source Ltd, Chilton, Didcot, Oxfordshire, OX11 ODE, UK Tel (direct): 01235 778664 -- From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Jacob Keller [j-kell...@fsm.northwestern.edu] Sent: 21 June 2012 17:11 To: ccp4bb Subject: Re: [ccp4bb] Detergent and protein oligomerization First of all, isn't the choice either dimer or trimer, and second, as a protein-detergent complex (PDC), it would be very unlikely that a trimer of 99 kD would run at 100 kD, although all is fair in love, war, and membrane proteins. JPK On Thu, Jun 21, 2012 at 10:50 AM, Raji Edayathumangalam r...@brandeis.edu mailto:r...@brandeis.edu wrote: Hi Everyone, Sorry for the non-CCP4 post. I have a very basic question about detergents, critical micelle concentration and behavior on gel filtration. A 33kDa membrane protein was purified by gel filtration in a buffer containing 0.4%(w/v) beta-NG (CMC: 6.5mM) and 0.046%(w/v)LDAO (CMC: 0.14mM). So the concentrations of beta-NG and LDAO in the gel-filtration buffer are ~2X and ~14X that of the CMCs of the respective detergents. The elution volume of the protein peak (plus detergent) on Superdex200 corresponds to a molecular mass of 100kDa. I think that the 100kDa mass above includes contributions from both the protein as well as the detergent micelles. If this is correct, is it then accurate to try to glean the oligomerization state of the protein (and conclude that it is a trimer or tetramer) without taking into account detergent micellar mass and its influence on elution volume? How should one interpret the 100kDa mass estimate from the gel filtration? Thanks. Raji -- Raji Edayathumangalam Instructor in Neurology, Harvard Medical School Research Associate, Brigham and Women's Hospital Visiting Research Scholar, Brandeis University -- *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program email: j-kell...@northwestern.edumailto:j-kell...@northwestern.edu *** -- This e-mail and any attachments may contain confidential, copyright and or privileged material, and are for the use of the intended addressee only. If you are not the intended addressee or an authorised recipient of the addressee please notify us of receipt by returning the e-mail and do not use, copy, retain, distribute or disclose the information in or attached to the e-mail. Any opinions expressed within this e-mail are those of the individual and not necessarily of Diamond Light Source Ltd. Diamond Light Source Ltd. cannot guarantee that this e-mail or any attachments are free from viruses and we cannot accept liability for any damage which you may sustain as a result of software viruses which may be transmitted in or with the message. Diamond Light Source Limited (company no. 4375679). Registered in England and Wales with its registered office at Diamond House, Harwell Science and Innovation Campus, Didcot, Oxfordshire, OX11 0DE, United Kingdom -- *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program email: j-kell...@northwestern.edu ***
