My questions/comments: * Do you plan to express the fusion proteins from plasmids for the localization studies? If the goal is to understand native localization, I would be cautious with overexpression and consider tagging the proteins at their endogenous chromosomal loci instead. * Would it be useful to first confirm the protein–protein interaction in vivo? For example, a split NanoLuc/NanoBiT approach could provide evidence that the interaction occurs inside the cell while minimizing the size of the tag. * Have you considered using a small split-GFP tag such as GFP11? This could potentially allow localization of the proteins with minimal perturbation. You could also consider localizing both proteins separately; if they are found in the same cellular compartment and the interaction is independently confirmed in vivo, that may provide a strong overall picture. * One point to keep in mind with GFP complementation approaches is that the reconstituted fluorescence can be stable and may not always represent the exact real-time location of the interaction event, so interpretation of the localization signal would need to be done carefully.
On Monday, July 06, 2026 06:53 CEST, Harshitha H N <[email protected]> wrote: I am designing a bacterial protein interaction and co-localisation study involving two interacting proteins. One protein is very small (~7–8 kDa), while the other is approximately 25–30 kDa. Previous biochemical experiments (crystal structure and binding studies) have confirmed that the two proteins interact, and one protein modulates the function of the other. My initial plan was to generate fluorescent protein fusions (EGFP and mCherry) for live-cell co-localisation. However, I am concerned that fusing a ~27–29 kDa fluorescent protein to the smaller protein may interfere with its folding, localisation, or interaction. ______________________________________________________________________________ To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1 ######################################################################## To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1 This message was issued to members of www.jiscmail.ac.uk/CCP4BB, a mailing list hosted by www.jiscmail.ac.uk, terms & conditions are available at https://www.jiscmail.ac.uk/policyandsecurity/
