Dear That was a quite enlightening discussion!! I am grateful to you guys for your time!! I will definitily try some of these to get a clear answer.
Regards Intekhab alam On Tue, Aug 10, 2010 at 8:38 AM, Bostjan Kobe <b.k...@uq.edu.au> wrote: > Dear Intekhab > > Let me just add to this that gel filtration is not an accurate method for > determination of molecular mass, because the migration on the column > depends > on the shape of the protein. > > The following methods can be used to determine molecular mass irrespective > of shape: > - MALLS (multi-angle laser light scattering or static light sxattering) > - sedimentation equilibrium on analytical ultracentrifuge (AUC) > - native mass spectrometry > > For a short recent review on issues associated with determining oligomeric > state from crystal structures, with older references and relevant > bioinformatic tools cited in there, please see > http://www.ncbi.nlm.nih.gov/pubmed/19021571 > > Bostjan > > > On 10/08/10 6:26 AM, "Maia Cherney" <ch...@ualberta.ca> wrote: > > > To determine the oligomeric state of a protein (monomer or dimer in your > > case), it's useful to use the PISA server. You upload your pdb file from > > the crystal structure.The server calculates the areas of interfaces > > (buried area) and deltaG (change in Gibbs energy) upon oligomer > > dissociation. (E. Krissinel and K. Henrick (2007). /Inference of > > macromolecular assemblies from crystalline state/. J. Mol. Biol. *372*, > > 774--797 . E. Krissinel and K. Henrick (2005). /Detection of Protein > > Assemblies in Crystals/. In: M.R. Berthold /et.al./ (Eds.): CompLife > > 2005, LNBI 3695, pp. 163--174 <http://dx.doi.org/10.1007/11560500_15>. > > E. Krissinel (2009). /Crystal contacts as nature's docking solutions/. > > J. Comp. Chem., in press; published on-line 6 May 2009; DOI > > 10.1002/jcc.21303} > > If the interface area (divided by 2 per one protomer) is greater than > > 1000 A2 and delta G is more than 5kcal/mol (the higher the better), it's > > a dimer. However, don't forget that most dimers can dissociate into > > monomers upon dilution. There is a dynamic equilibrium between dimers > > (oligomers) and monomers that depends on their concentration and the > Kdiss. > > Separating them in any method will disturb this equilibrium. If the > > re-equilibration time is greater than the separation time, you can see > > both monomers and dimers. You can even roughly calculate the > > dissociation constant: > > > > Kdiss=[monomer]2/[dimer] where brackets mean concentrations. To give you > > an estimate, at Kdiss=10(-3)M, you have roughly equal concentration of > > dimers and monomers at 10-3 M and only 10% dimers at 10-4 M. Sometimes, > > protein needs to dissociate easily for the biological function. > > > > Maia > > > > intekhab alam wrote: > >> Hi everyone > >> Sorry for some non specific query!!!!! > >> > >> i am working with a protein that shows a dimer in the crystal > >> structure but when i tried to figure out that with standard molecular > >> markers in gel filteration (superdex-200, 24ml column) it turned out > >> to be a monnomer. Native gel analysis after incubating the protein at > >> 20 degree, 37 degree showed more dimer at 20 degree celcius as > >> compared to 37. I tried similar strategy in gel filteration by > >> incubating my protein at various temperature,where a lot of > >> precipitation was observed at 37 degree celcius and after removing the > >> precipitates i run the gel filteration that has 0.5 ml higher elution > >> volume as compared to samples incubated at 20 degree celcius and 4 > >> degree celcius.( Is this significant) > >> Furthermore i have done some experiments in cold room (4 degree) where > >> the elution volume is stuck at a point irrespective of the conditions > >> (as Flow rate, concentration of protein etc) and that is higher than > >> that of the room temperature by 1 ml. > >> Standard moleculr weight markers also show higher elution volume in > >> cold room in comparison to the room temperature by 1 ml. > >> > >> I will be highly obliged if someone suggest some literature or any > >> otherway to do gel filtrtaion so that i can clearly resolve this > >> issue. Also let me know if there is some literature > >> available on effect of temperature on the elution volume of proteins. > >> > >> Thanks in advance > >> > >> -- > >> INTEKHAB ALAM > >> LABORATORY OF STRUCTURAL BIOINFORMATICS > >> KOREA UNIVERSITY, SEOUL > > --- > Bostjan Kobe > ARC Federation Fellow > Professor of Structural Biology > School of Chemistry and Molecular Biosciences > > and Institute for Molecular Bioscience (Division of Chemistry and > Structural > > Biology) and Centre for Infectious Disease Research > Cooper Road > University of Queensland > Brisbane, Queensland 4072 > Australia > Phone: +61 7 3365 2132 > Fax: +61 7 3365 4699 > E-mail: b.k...@uq.edu.au > URL: http://profiles.bacs.uq.edu.au/Bostjan.Kobe.html > Office: Building 76 Room 329 > Notice: If you receive this e-mail by mistake, please notify me, and do not > make any use of its contents. I do not waive any privilege, confidentiality > or copyright associated with it. Unless stated otherwise, this e-mail > represents only the views of the Sender and not the views of The University > of Queensland. > > > > -- INTEKHAB ALAM LABORATORY OF STRUCTURAL BIOINFORMATICS KOREA UNIVERSITY, SEOUL
