Dear Researchers,
Currently I am struggling with generation of stable mammalian cell line expressing my protein of interest (POI) fused to NanoLuciferase. Unfortunately, when I run Western Blot on whole cell lysates I see expression of not only my fusion protein but also free NanoLuc which are on approximately the same levels. Do you have any ideas why could it be that way?
Thanks in advance for any hints!
Hi Malwina
I can think of 3 possibilities:
1. A protease is cutting your fusion protein. You can check for recognition sequences using ExPASy or MEROPS.
2. Your fusion protein is unstable and breaks apart. Can you detect your POI without the luciferase attached to it in your samples? If so, changing the linker sequence might help to improve protein stability. I recommend "Fusion Protein Linkers: Property, Design and Functionality" by Cheng et al. for a nice overview of what you can use instead.
3. The DNA you are using for transduction is contaminated with luciferase expression vector. But I'm sure you would have noticed that already.
I hope this was at least a bit helpful. Good luck on your experiments!
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