How to insert a Tag to the middle of a protein?
Posted 08 December 2004 - 07:51 AM
I am going to insert a tag(about 10AA) into the middle of a protein. Since connectting tag to the N or C-terminal were proved interfering the function of the protein. It is a protein about 400aa. I have no idea how I can do that. Who can give me some suggestion. As I know, PCR probably can do that, but I don't know how it work. Any suggestion will be appreciated.
Daniel from UNL
Posted 08 December 2004 - 09:03 AM
Hope this will help a little bit...
Posted 08 December 2004 - 09:52 AM
(3) detection of the tag will be more difficult...
You need to be careful regarding where to put the tag if this is really necessary. Two approaches you can try: (1) PCR 2 fragments first (the N part with the tag and the C part), then ligate 2 together, be careful to be sure it is in-frame after ligation; (2) custom gene synthesis if money is not the problem. Our lab has good relation with Epoch Biolabs' service and they are one of the pioneers in this field, check thyem out (~$1.4-1.5 per bp).
Posted 08 December 2004 - 03:37 PM