I want to use a plasmid that is HA tagged. My research is in vitro and I am bit confused on what to do. Thank you!
Hi there,
Transfection efficiency is the ratio between cells expressing the transfected protein and the total number of cells. So in your case it will be about detecting HA epitope (by IM I guess) and counting cells.
Hi,
Give more details, HA tags are for in vivo or cellular expression.
HA antibodies work pretty well.
Good luck...
Anand Patwardhan Dominique Liger Thank you for responding. I want to transfect the T98G cell line with a plasmid from a pseudogene. However, the plasmid description on Addgene mentioned that there is a HA (N terminal on backbone) tag. I was wondering if there is any way to get the transfection efficiency using this tagged protein, without the HA antibody would be preferable. Again, thank you so much.
Hi Sammy,
Firstly, if the tag is N-terminal, you need to ensure you remove your ATG start codon off the front of your sequence you want to express, otherwise there's a chance you'll get un-tagged proteins.
Secondly, your description about what you want to actually do is a bit confusing... do you want to insert a sequence of interest into the plasmid for expression in your T98Gs? Or does the plasmid already contain a pseudogene that you want to express?
HA tag is quite a nice tag, I've used this previously for expressed proteins both for protein expression and transfection. In order to calculate transfection efficiency, you'll need an anti-HA antibody. Grow your cells on coverslips, transfect, fix (I use 3-4% PFA) and then do IF on it - you can use either dapi/hoescht to show nucleus or phalloidin to show actin. You then just count your cells that show HA labelling and work out the ratio of labelled against unlabelled to show your efficiency.
Alternatively, you could always label your protein directly if you have an antibody against that? This way you don't have to use HA antibody...
However, short of labelling your cells and doing IF... there is no other way (that I can think of) that you can work out transfection efficiency... bar maybe expressing a GFP/YFP/RFP-tagged construct?
Hope it helps!
John
- Badges
- Science method