Is it good to use protein tagged with fluorescent proteins or other kind of probes?
Hi Chandhuru, an easier approach is to use Life Technologies ER Tracker dyes. These can be used for microscopy or Flow Cytometry. You load the cells for 30 min at 37C at 100nM for FACS and more for microscopy. These dyes can be used semi-quantitatively, see my website for estimations of ER-phagy link http://www.icms.qmul.ac.uk/flowcytometry/uses/autophagy/organellephagy/index.html
Gary
Antibodies against BiP, PDI, Calnexin etc can be used as a marker for ER in HeLa cells.
Both Gary and Anirban have great suggestions. The key issues are why you want to track the ER (not sure exactly what that means), and what kind of experiments are you planning on?
Hi Neil, I want to check whether my protein is localized in ER or in some other organelles. TO check this I want an ER tracker.
That said, I think I would go for co-staining with any of the antibodies Anirban suggested (provided there is species cross reactivity with the cell you are using). However, I would caution against using co-staining as the sole piece of evidence if this is really crucial to your studies. You will have to do additional approaches such as Optiprep gradients to provide parallel evidence.
Hi Chandhuru, I have used PDI, BiP and calnexin antibodies for staining ER in HeLa cells. What I have noticed, is that theses markers stain different parts of the ER, therefore you might find that your protein of interest colocalise with one, but not with the rest of the markers, so I would go for testing at least two or three of them. And still, this will be only an indication, that your protein of interest might be in the ER. What I also would use, some negative controls: f.e. stain also with markers for Golgi or endosomes. These should not colocalize (at least not to a big extent) with ER markers (this way you will see if your staining is somewhat specific for ER). Then if possible, use some negative control for you protein of interest (or some treatment that is expected to change its localisation). Good luck!