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" <[email protected]> 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: [email protected] 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.
