Hello Theresa, Maybe a functional assay (if possible) is better than determining the oligomeric state? Why is finding out the oligomeric state (which is unknown anyway and the answer can be compared to nothing), the answer to "is having a tag ok?" "will the protein crystallize"? What if the analysis shows a mixture of different protein oligomeric states in solution? For the general analysis, try SEC-MALLS. I guess the accuracy is 90-95 % for membrane proteins. You might have however to try different detergents or try different columns depending on the empty micelle size and how close it is to the protein-micelle peak etc (please ask if you need a detailed explanation). You will still need to do a func assay, because again you don't know if a particular detergent is harsh from the beginning affecting the oligomeric state, and it is not the tag. Best wishes toufic
PS: (1) 100 % accuracy is a complicated term (even crystallography has a R-factor). The error value might be a different oligomeric state or simply an error of the method or experiment. (2) there is always a problem with detergents and lipids with memb proteins :) (3) Thermofluor is just a method, might need fluorescence background optimization, also trying different hydrophobic compounds/detergents etc ****************************************************** Toufic El Arnaout Trinity Biomedical Science Institute (TCD) 152-160 Pearse Street, Dublin 2, Ireland ****************************************************** On Thu, Mar 21, 2013 at 6:10 PM, Theresa Hsu <theresah...@live.com> wrote: > Dear all > > I have a His-tagged membrane protein with unknown oligomerization state. > But I am worried that tag addition may induce different state than in > native and affect its crystallizability. > > Is there a single method that can determine the oligomerization state with > nearly 100% accuracy? I have use of AUC and SAXS but there seems to be > ambiguity about detergent and lipid effects. Is Thermofluor a right method? > > Does oligomerization require special assembly proteins, which will mean > that tag cleavage is not useful to obtain native state? > > Thank you. > > Theresa > > >
