Cell culture for immuno-precipitation

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adagnall's picture
Cell culture for immuno-precipitation

I want to start a new experiment to confirm results I obtained using a bacterial 2 hybrid system, which identified putative interactants of my gene of interest. Seemingly confirmation is done by immuno-precipitating your gene of interest with its putative interactant after stably expressing both in a eukaryotic cell line.

I have never done cell culture before. Can anyone help suggesting which cell lines would be best for this procedure? For instance: suspension or monolayer, which system is best to transfect, are certain cell lines better at expressing protein etc?

macbride's picture
That's a pretty broad

That's a pretty broad question. If you've never done cell culture before, you really need some hands-on tutorial from an experienced cell culturist. You also need a fair amount of specialized equipment and reagents. If you only need to one experiment, you are better off having someone else do it for you. Cell culture is too tricky to just try for one experiment, in my opinion. That said, I prefer adherent cells (much easier to wash and change media, and at least in comparison to the suspension cell lines I've worked with, they are infinitely easier to transfect). Your choice of particular cell line will depend on a few things: for instance, if you were studying a gene overexpressed in breast cancer, it might make sense to choose a breast carcinoma cell line. Or your choice might be limited by what cell lines you can get from your institution or other labs. In terms of generic, easy to maintain and transfect cell lines, I like CHO and CV-1, but there are lots of others that people use quite happily.