If the protein is eukaryotic you may need to express it in eukaryotic cells, probably with a tag for later identification / purification of the fragments. This might work for a plasma membrane protein, -but if the protein is from an internal organelle then to find the correct topology you may need to purify the organelle. Proteolysis is easy to apply, -several proteases from the outside of the cell followed by mass spec of the species with the tag. mutagenesis and chemical labelling is another approach. If the protein has a cleaved signal sequence that would give a strong experimental clue to sidelines also. Robert Stroud str...@msg.ucsf.edu
> On Dec 10, 2017, at 6:33 PM, Firdous Tarique <kahkashantari...@gmail.com> > wrote: > > Hello everyone > > I am trying to clone some domains of a transmembrane protein. I have used an > online server "Phobius" for the prediction of sequences which is showing it > to be a membrane protein with cytoplasmic, non cytoplasmic and transmembrane > regions. Attached is the image of that prediction. Regions from 100-200 amino > acid residues which I am more concerned about is predicted to be cytoplasmic > in nature while other servers have predicted it to be non cytoplasmic in > nature. I am having problems in expressing this domain. Sometimes the > prediction may go wrong for such a short stretch of amino acid residues. > > My question is to know about any technique or any classical experiment which > can actually tell me the exact location of this domain across the membrane. I > mean real experiments not predictions. > > Regards > > Firdous > Graduate student > Delhi University > India > <p1c11le9sd1o59vndpqe17c28nb4.png>
