His-tag design in primer - DO i need filler. (Nov/28/2005 )
Designing a histag infront of my insert (dont want to cut it off) and was wondering if I need bases between the his-tag sequence and the insert?? if not...then why do so many vectors possess such fluff.
Regards,
John
I'm not entirely sure of your question, but remember that a functional fusion requires not only cloning the insert downstream of the his tag, but maintaining translation frame. Are the extra bases you're talking about there to allow cloning into the correct frame?
hi
i think as homebrew that extra bases are for cloning and ensure that the restricion sites provided are to maintain the in frame coding.
i think as homebrew that extra bases are for cloning and ensure that the restricion sites provided are to maintain the in frame coding.
Hmm..Well its like this ...We have a restriction site, a start codone,,,the histag sequence and then the insert fused. Thanks for your replies.
John
Hi
The think is you usually put between a tag and a protein some amino acides (for example 3xG) they are called LINKER. This is to avoid kind of 3D changes in the protein because of the tag inserted. Imagine for example that the end of your protein is located inside the protein; if you add the tag directly after the protein you for sure will be changing the 3D of the prot. This give also kind of flexibility between the protein and the tag.
I hope I helped you.
One advice: I will allway try bevor with a linker.
cuca