I am looking for an article that explains which is the protein target for dsRed transfected by lipofectamine in mice cortical neurons.
Is dsRed fused to a protein in particular? Or is paired randomly?
Thank you for your information! That help me to understood what is happening to my experiments. About the transfection with lipofectamine, has been hard : S but finally we have set up succesfully a protocol. If you need it, please do not hesitate to contact me.
Hi Fernando! I recommend that you check in the litterature first if your protein could be fused with a fluorescent tag N- or C-ter then you can use a Clontech plasmid. You can also check in addgene http://www.addgene.org/, they have lot of published plasmids and they verify the sequence. Be careful though, all red fluorescent tag do aggregate more easily and could lead to an artifact in the protein localization. You can do a simple test experiment in cultured cells if you have an antibody against your protein to compare the localization with the endogenous. Also you can transfect the red construct with a another one (GFP, YFP...) and ask if they do colocalize. Alternatively, you can use cerulean tag (blue) if you're able to detect it with your microscope, it's not bleaching like the CFP, it's bright and doesn't have this aggregation pb. For the neuronal transfection wit lipofectamine the trick is to have an healthy culture, and transfect no later than DIV 11 and for 3h top! But I agree that lentivirus infection is more efficient. I'm sending my paper in couple of weeks, it's mainly live imaging on hippocampal neurons you can check it out when it'll be published ;)
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