Our protocol allows to identify and analyze interactions between multi transmembrane proteins of the endoplasmic reticulum and golgi complex in living cells at low expression levels. The main advantage of this technique is it's to special sensitivity, which allows to avoid high over expression levels that are often detrimental to cellular health and may disturb protein subcellular localization. Harvest the adherent HEK293 T-cell culture by trypsinization and re-suspend the cells in a dedicated complete growth medium.
Plate the cells onto a clear bottom, white side 96-well plate. Adjust the total number of wells to accommodate all tested combinations and controls, including replicates. Then, culture the cells overnight in standard conditions.
On the next day, transfect the cells with the desired combinations of expression plasmids. Dilute the plasmids in a serum-free medium to 6.25 nanograms per microliter for each construct and add the lipid-based transfection reagent. Add an appropriate lipid to DNA ratio.
Incubate the plasmids according to manufacturer's instruction. Then, add eight microliters of the lipid DNA mixture to the designated wells. Mix the content of the plate with gentle rotation and culture the cells for 20 24 hours in standard conditions.
On the next day, replace the conditioned medium in each well with 100 microliters of a serum-free medium, making sure that the cells do not detach upon medium exchange. Just before measuring luminescence, mix one volume of furimazine with 19 volumes of a dilution buffer. Add 25 microliters of the furimazine working solution into each well, and gently mix the plate by hand or with a orbital shaker.
Insert the plate into a luminescence micro plate reader and equilibrate the plate at 37 degrees Celsius for 10 15 minutes. Select the wells to be analyzed and read luminescence with an integration time of 0.3 seconds. Continue to monitor luminescence for up to two hours when required.
This protocol was tested on two nucleotide sugar transporters, SLC35A2 and SLC35A3, which are golgi-resident type three membrane proteins with N and C termini facing the cytoplasm. There were eight possible tagging options and the resulting fusion proteins could be set together in eight possible ways. Mean relative luminescence units or RLU values corresponding to all the tested and control combinations are shown here.
The RLU values obtained for three of the combinations were significantly higher than those obtained for the corresponding control combination. To obtain ratio values for the combinations of interest, the RLU values were divided by the RLU values obtained for the respective controls. Ratios between 10 and 1000 are highly indicative of specific interactions.
There were two such combinations supporting the idea that SLC35A2 and SLC35A3 interact. To confirm this data, HA tagged SLC35A2 or SLC35A3 were co-expressed with the combination that resulted in the highest relative luminescence. Varying amounts of the plasmids and coding HA tagged NST variants were used for co-transfection, resulting in a statistically significant dose dependent decrease in RLU values.
The most critical thing is to tackle your protein self interest in all possible ways and to assay all possible combination, as some tagging orientation may produce false negative results. Once the best performing combination has been selected, the dynamics of the corresponding interaction can be monitored to, for example identify agents or conditions that disrupt your complex of interest over time.
