I've just started my PhD and am wanting to search for a construct on Addgene that expresses a tagged version of my protein of interest. It's really just a preliminary search to see what constructs there are out there and see if there are any suitable for my experiments. I've never had to search for a plasmid construct before so I'm unsure what are the most important considerations and most effective ways to find what you want. Any help would be really appreciated.
Many factors of course but IMO there are 2 that will bite you in the ass down the road if you don't plan ahead. First, a promoter which won't be suppressed in your cell type of interest. Second, be sure to pre-screen your cell lysates/proteins with your anti-tag antibody before you decide to clone it into your construct. You don't want to find out later that you get 8 similar bands when running a western and your not sure which ones are your protein, or degradation products. And which are unfortunate cross-reactive proteins from your cell type of interest. With a quick pre-screen you can find a tag-type which is optimal for your fusion construct in the cell type your interested in using. Be sure to try to perform an assay identical to the ones your thinking of using down the road. IP, fixed confocal staining, western, and the like. If you run into issues then think about using a different tag when assembling your construct.
I'm sure you'll get more useful comments here as well. Good luck.
I agree there are many factors to consider depending on your experiment. Not to repeat the great advice already provided, I would first suggest making sure that the vector can be used for the experiment you are interested in. For example, you would want a different plasmid if you want to express and purify protein from bacteria (ex. IPTG inducible construct with a T7 promoter) versus expression in mammalian cells (ex. mammalian promoter in a lentiviral or retroviral vector to make a stable cell line). The second factor is what you need the tag for to help select which tag to use. Again, if you want to purify the protein from bacteria you may want a His tag to purify on a nickel column whereas if you want to see the subcellular localization in mammalian cells you may want to use a GFP tag. Finally, I would also suggest to check out DNASU http://dnasu.org/DNASU/Home.do as another possible source of plasmids. They have genes in a number of different vectors for many different applications.
Best of luck with your experiments!
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