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.edu<mailto: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 *******************************************
