I have two proteins one is predicted to be type 1 transmembrane protein and the other is predicted to be type 2 transmembrane protein. I need to know how can i proof this experimentally
Thanks
A type 1 membrane protein has a single transmembrane span. In many cases, expressing a version of the protein lacking the transmembrane span results in a soluble protein. This would not work with a polytopic membrane protein.
@Adam
Type 2 transmembrane proteins also has a single transmembrane span but their orientation is different where their C terminus is extra-cellular while type 1 have their N terminus extra-cellular.
I was thinking of tagging the proteins on their exposed terminus and after expressing them in cells I treat the cells with proteinase K and then lyse the cells and blot for the tags.
What do you think of this or do you have other ways?
Thanks
It sounds like a good idea. It would be useful to have a control antibody that recognized another part of the protein that would be present in both cases.
Hi there,
Do you intend to express the protein in bacteria ? If so, by constructing a fusion protein with BLA (at Cter of your protein sequence) and express the fusion in bacteria, phenotype towards ampicillin will tell you about Cter location : if it's intracellular, the cell will be sensitive to ampicillin, if it's extracellular(periplasmic) cell will be resistant.
If expressing the protein at eucaryotic cell surface, a fusion with an easily detected epitope and immunofluorescence on intact cells will tell you about topology.
Hi Salim,
I can quickly think of 2 ways:
1) You can reconstitute the portein in vesicles, and eslecifically attach a small fluorescent molecule to either the C-terminus or N-terminus (there are fluorophores reactive to either the N-terminus, or alternatively cysteines or lysines you could replace there). Then you can probe its accesibility from the cis-side of the membrane with a quencher (KI, for -example). A tag in the cis-side will have a more efficient quencher than one in the trans side. The following reference deal with determination of topology using a similar approach (fluorophore quencher). Their approach is a little bit more complex because they are trying to achieve other things as well, but you can get the idea. http://www.sciencedirect.com/science/article/pii/S0003269705007591
2) You can attach a short peptide/tag to either side of the protein (N- or C-terminus) and leave a proteolytic site in the link connecting your tag with the protein. Then you can probe the accesibility to either side with the protease (trapping the protease inside vesicles for example). Couple of references expanding in the idea:
http://pubs.acs.org/doi/abs/10.1021/cb500388m
http://www.sciencedirect.com/science/article/pii/S0005273614003289
Good luck!
Hi Salim, are these proteins localized at the plasma membrane of cells you use to express them? If so, you may try adding a tag like HA to the C-terminus of the coding secuence of your proteins. Then express the tagged proteins and incubate cells with an antibody to HA. You should see staining only for type 2 protein as the tag would be extracellular but not for type 1. The tag for type 1 protein would be accessible to antibodies only after permeabilization. Additionally you can try adding the tag on the N-terminus instead of the C-terminus and you should expect the opposite results.
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