Should I check expression of my LgBit and SmBit fusions by Western blot?

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  • Last Post 21 May 2018
MARKBOND posted this 04 May 2017

Hi

I am currently cloning my genes into the SmBIT and LgBIT vectors. I am just wondering if I should check expression from these vectors by Western blot before looking for luminescence off the interaction? If so, are there any LgBIT and SmBIT specific antibodies?

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alandrem posted this 05 May 2017

Hello @MARKBOND. It isn’t necessarily required to check for the expression of full-length fusion proteins. We don't do this every time that we develop a new assay. Typically we go right into our optimization experiments of testing the different combinations in order to find the best performing pair using the strategy outlined in section 3 of the tech manual. Can you share a little about the assay you are developing? Do you have a tool compound or known modulator?

However, we do know that many researchers would like to confirm that they are expressing the full-length fusion to ensure that nothing unusual happened during the cloning. Especially if any troubleshooting is required during assay development. If you have primary antibodies against your proteins you will see a size shift for the LgBiT fusion, but the SmBiT fusion can be difficult to distinguish . We are currently working on antibody development. We do not yet have an antibody for SmBiT. However, we do have a polyclonal raised against NanoLuc that also recognizes LgBiT. Would you like an aliquot of this? Just let me know and we can get one out to you. Or let us know if there is additional support we can provide.

jianny115 posted this 02 March 2018

Hello, I am using NanoBiT PPI system in HEK293 cells.  My target proteins are membrane proteins. It is difficult to predict if tags would alter their localization. I'd like to check it. Because there are endogenous expression of my target protein in HEK293, antibodies against them would not work. It would be great if  I can get an aliquot of antibody against NanoLuc that also recognizes LgBit.  Could you ship an aliquot to me? Thanks.

alandrem posted this 16 March 2018

Hi Jianny115, Sorry I missed your post! Yes, we have an anti-NanoLuc polyclonal that also recognizes LgBiT. Yep, I will message you separately to get the aliquot out. Thanks!

sanmarco96 posted this 21 May 2018

Hello, I am also using nanoBIT PPI system in HEK293 cell lines. I also would want to use anti-NanoLuc antibody recognize LgBit as well as SmBit. I hope you contact me soon. Thank you!

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