Hi folks,
I'd like to know how can I detect whether a protein has been incorporated into a liposome, and how much of it is entered the vesicle.
What kind of experiment can i perform?
thank you very much.
Hi there,
Monitor the sensitivity to exogenously added protease (proteinase K) seems the simplest experiment to run. You first have to make sure that the treatment is not damaging the liposomes. The protein having entered the liposome will become resistant to protease treatment.
Hope this helps.
Hi there,
Monitor the sensitivity to exogenously added protease (proteinase K) seems the simplest experiment to run. You first have to make sure that the treatment is not damaging the liposomes. The protein having entered the liposome will become resistant to protease treatment.
Hope this helps.
An excellent suggestion from Dominique Liger. Another method is to pellet the liposomes by ultracentrifugation and see how much of the protein is in the pellet, compared with a solution of the protein alone. A third method is to pass the liposomes through a gel filtration column that would separate liposomes from the unicorporated protein and see how much of the protein coelutes with the liposomes. A fourth method is to attach a fluorescent dye to the protein and test for the sensitivity of the dye to a quencher.
yes ok, but how can i distinguish a protein that has entered the liposome from another that interacts on the surface of liposome? In other worlds with centrifugation and gel filtration, i can separate the protein bound to liposome, but how can I know weather the protein is interacting on the surface of the liposome or it has enetered the vescicle?
concerning the use of proteasis, i'll take it in account, but at the moment i prefere alternative methods.
The proteolysis and fluorescence quenching methods will distinguish external bound protein from internal protein in sealed vesicles.
@ Adam B Shapiro please send me any reference paper regarding fluorescence quenching for the determination of external bound protein from internal proten in sealed vesicles.
This paper sounds like it might have a good example:
http://www.ncbi.nlm.nih.gov/pubmed/3893545
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