On 11 Jul 1997, Luc CAMOIN wrote:
> Hi,
>> I'am looking advice about protein tag.
>> Is it better to put the tag on C-terminal instead than N-terminal?
>> Which tags are the best for detection in western blot?
>> Which tags are the best for affinity purification of recombinant protein?
>> thanks in advance
>> Luc CAMOIN
>>>>If you put the tag on the c-terminal end of the protein, you will only
get signal if the entire protein is there, since transcription is N-->C.
However, the c-terminal of some proteins is buried within the structure,
so the tag may not be apparent to antibody, or it may disrupt the
three-dimensional structure of the protein and ruin it's activity.
We commonly use the HA tag in our lab for wetern detection, and we use
6His tags on our recombinant proteins for purification with Ni/NTA
agarose. Invitrogen makes vectors for both tags, and we use Qiagen Ni/NTA
affinity gel. Incidentally, I prefer batch purification vs. column
with the affinity resin. I get lower background and better adsorption of
the target protein.
Good luck and have fun.
-John Notis
