HA tag vs His(6) tag , with mutagenesis - (Jul/22/2006 )
I would like to introduce a tag at the 3' position of my cDNA, cloned into a vector (for immunolocalization). I can't use the restriction sites of vectors with tags of HA and histidines, and instead of creating the restriction sites by PCR amplification, I'd like to use the Quickchange XL site-directed mutagenesis kit and create an "insertion" disrupting the stop codon of my cDNA, and creating the epitope for HA or six histidines.
I don't know if this is a good idea. It would be a large insertion (althought some people have created 80 pb deletions with this kit with great efficiency) and I don't know if it's going to work.
Moreover, I've heard that the his (6) tag doesn't work very well, and it's better to use the HA tag with an anti-HA antibody.
I need a little piece of advice about this issue. It is a good idea to create the epitope by mutagenesis? Which is better, hemaglutinine or polyhistidines?
if u insert HA tag how u purify the protein?
I don't need to purify my protein, as I want the HA for western and immunofluorescence (detection with antibodies only, not purification)...
Sequence ur new construct to verify if the tag is fine.
Yes, sure I'll do it, but do you think the idea of mutagenesis is a good one?
I've heard of people doing that. You could give it a try but I prefer to amplyfy the tag from another plasmid and cloning it the usual way. Even digesting the tag from another plasmid would be fine. Whenever I can, I prefer the traditional cloning without PCR because of possible mutations
in this given case i would go for HA tag, coz it is more immunogenic than His tag. because of this reason u will have good antibody and work will be done in much simpler way.