Hi Yong-Heng,

Split GFP is used to study protein interactions at the cellular level in an experiment called BiFC for Bimolecular Fluorescence Complementation.

Split GFP is unable to form a complete, stable, fluorescent protein spontaneously. You have to force the reassociation of the total GFP by fusing each half with interacting proteins.

Your question suggests that you have no interacting proteins, only GFP halves. If so it is perfectly normal that you have no fluorescence.

If you have fused with interacting proteins, have you made a positive control ? With alpha tubulin fused to GFP1-10 in Ct, and beta-tubulin fused with GFP1-11 in Nt and all possible combinations with alpha/beta, Ct/Nt and 1-10/1-11 for example. Have you done all the possible combinations replacing tubulins with your target proteins ?

If you have fused with GFP halves, and you know that your proteins interact in vitro (by GST pulldown or CoIP), in vivo (2-hybrids), but you are unable to see fluorescence while you positive controls are ... positive, then you can say that, in your system, the two proteins don't interact. You have to control by spectrofluorimetry that in vitro your BiFC is functionnal.

Hope this helps you

Cheers

Oliv

Dear Olivier,

thank you very much for the information. I fused two proteins known to form complex in cell to the splits respectively and the un-fused halves as negative CK, both of which have no signal. I will try the positive CK you mentioned to confirm again.

thanks a gain for your help ^_^

Cheers,

Yong-Heng

Yong-Heng, if you haven't tried yet, you may try to change the orientation (split A on C-terminus or N-terminus and Split B on C-terminus or N-terminus) of your fusion proteins and transfect different combinations. In total you have 4 different constructs. For steric reasons and confirmation state your splits may not complement If not close enough.

Good luck!