Tag proteins for CO-IP and confocal - (May/06/2011 )
I am planning to start some experiments for protein-protein interactions using confocal and CO-IP
I want to construct plasmids labelled with either GFP and His. I will use the His tag for IP.
I am going to use cDNA using primers with restriction site extensions to clone into plasmids. The His Tag is at the N terminus and I will also probably get a GFP plasmid tagged at the N terminus. The cDNA will be inserted inframe following the Tag, so does it need to include the start codon? I assume that it has. If the tag had to be at the C terminus and so the MCS is before it, do I have to include the inserted gene's stop codon? Or if I do the tag will not be fused with my protein?
If you tag C-terminally do not include the gene's stop codon or you won't get the tag expressed.
I include the start codon at the N-terminus even if there is one on the tag. You can add a kozak sequence before the start codon to enhance the expression. This should take the form GCCACCATG, where the ATG is the start codon.