Fla 3000 laser fluorescence scanner

Competitive ABPP using HEK293 cell lysates and mouse brain membranes was conducted to visualize effects of selected triterpenoids on the binding of the active site serine-targeting fluorescent fluorophosphonate probe TAMRA-FP following outlines of previous protocols [4] (link), [31] (link). Briefly, lysates (25 µg) of HEK293 cells with or without (Mock) hABHD12 overexpression or mouse wholebrain membranes (100 µg) were treated for 1 h with DMSO or the inhibitor, after which TAMRA-FP labelling was conducted for 5 min (lysates) or 1 hour (membranes) at RT (final probe concentration 2 µM). The reaction was quenched by addition of gel loading buffer (followed by boiling for 5 min in the case of lysates), after which 10 µg protein (10 µl) was loaded per lane and the proteins were resolved in 10% SDS-PAGE together with standards. TAMRA-FP labeling was visualized (λ_ex 552; λ_em 575 nm) by a fluorescent reader (FLA-3000 laser fluorescence scanner, Fujifilm, Tokyo, Japan). The intensity of bands was quantified using ImageJ, a freely available image analysis software (http://rsbweb.nih.gov/ij/).

Competitive ABPP using mouse whole brain membranes or HEK293 cell lysates was conducted to visualize the effect of selected inhibitors on BAT5 activity in native brain membrane proteome and in lysates of HEK293 cells with transient expression of hBAT5 or mBAT5. We used the active site serine-targeting fluorescent fluorophosphonate probe TAMRA-FP as previously described [10] (link). Briefly, lysates (25 µg) of HEK293 cells with or without BAT5 overexpression or whole mouse brain membranes (100 µg) were treated for 1 h with DMSO or the selected inhibitors, after which TAMRA-FP labelling was conducted for 1 hour at RT (final probe concentration 2 µM). The reaction was quenched by addition of 2xgel loading buffer, after which 10 µg protein (10 µl) was loaded per lane and the proteins were resolved in 10% SDS-PAGE together with molecular weight standards. TAMRA-FP labeling was visualized (λ_ex 552; λ_em 575 nm) by a fluorescent reader (FLA-3000 laser fluorescence scanner, Fujifilm, Tokyo, Japan). The intensity of bands was quantified using ImageJ, a freely available image analysis software (http://rsbweb.nih.gov/ij/).