Dear All,
I have a protein of interest that is fused to GFP at its C-terminus. I would like to add a HA tag at the N-terminus, and was wondering what strategy to use. Should I just introduce the HA tag by PCR and re-clone the PCR product into the GFP vector, or would it be easier to open up the construct and anneal an adaptor containing the HA sequence? Also what are your views on HA v. FLAG? I plan to express the fusion protein in mammalian cells and use the N-terminal tag to detect the fusion protein at the cell surface.
Using site-directed insertion/deletion as described http://bmcbiotechnol.biomedcentral.com/articles/10.1186/1472-6750-8-91 is the simplest way to insert these tags. You only need one mutagenesis PCR step to get this done. For cell surface detection, HA tag is better than Flag tag due to its less polarized amino acids.
Hi,
I've tried both strategies you are proposing in my lab with success. Each of the two strategies will give you the result you need without problems I think. I would suggest you to try first the annealing option essentially because it's fast and simpler than re-clone your protein. Make sure you have 2 appropriate restriction enzymes to achieve the correct orientation of your adaptor. Make sure also that your adaptor sequence have the correct translation frame of your protein (remember to accurately check the restriction enzymes sites) and a start codon. If your construct have the right restriction enzyme sites this strategy is the faster, in my opinion.
There's no particulary differences between Myc, FLAG or HA tags in my opinion, your application mainly depends on the quality of the antibodies that you can buy. Just pay attention to not alter the function or the localization of your protein. that's because myc, FLAG and HA are charged tags that may alter some functions of your protein. You may consider other tags such V5 for example. Your best bet is to see what groups working with similar proteins to those you want to study use for their own work, and start from there.
You wish to detect the protein at the cell surface. Is it a transmembrane protein or secreted? If so, you need to be careful of the N-terminal signal peptide that is needed for translocation to the cell membrane.
Both HA and flag work well. There are good mouse monoclonals to each tag. Just be careful when messing with the N-terminus of a protein that is trafficked to he membrane otherwise you may alter its cellular localization.
I've seen reports of deterioration in the quality of anti-FLAG monoclonals in recent years, and have heard from people with poor experience using anti-FLAG antibodies for various applications. In my hands, the 12CA5 anti-HA monoclonal as well as a rabbit polyclonal from Abcam both worked very well.
Andrea makes an excellent point about the possible modification of your protein's function by charged tags such as FLAG, HA, and myc. I have first-hand experience with this as well.
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