LumiPK

Guide (The following is our recommended protocol. This protocol is for guidance only and should be modified according to your specific needs).
1. Stock Solution Preparation
1.1 Solvent: DMSO.
1.2 Concentration recommendation: 10 μM.

2. Working Solution Preparation
2.1 Diluent: tris-HCl buffer; Opti-MEM (1% FBS); cell culture medium.
2.2 Working concentration: 1 μM; 10 nM; 20 nM; 40 nM; 50 nM.
2.3 Note: Adjust working solution concentration as needed; prepare fresh before use.

3. Staining Procedure
3.1 Sample type:[1]
3.1.1 Adherent cells: HEK293 cells.
3.1.2 Recombinant protein: PKLwt, PKLNluc.
3.2 Incubation conditions:
3.2.1 For live adherent cells: 1 μM working concentration, 37°C, 5% CO2, incubation time up to 60 min.
3.2.2 For recombinant protein: 10-50 nM working concentration, ambient temperature, 30 min incubation for fluorescence competition assays; 0.15-10 μM working concentration, room temperature, 1-2 h incubation for native PAGE.
3.2.3 For cellular NanoBRET assays: 10-400 nM working concentration, 37°C, 2 h incubation.
3.3 Washing steps:
3.3.1 For cell lysate activity assays: Wash cells after 48 h compound incubation prior to lysis.

4. Controls
4.1 Negative control: HEK293 cells treated with DMSO alone; HEK293 cells transfected with Nluc-only plasmid.
4.2 Competition control: HEK293 cells or recombinant protein treated with saturating concentrations of mitapivat (20 μM) to displace LumiPK.
4.3 Positive control: Known PKL activators (mitapivat, TEPP-46, DASA-58) for competition assays.

5. Detection & Analysis
5.1 Instrument:
5.1.1 Confocal fluorescence microscope: Ex = 488 nm,.
5.1.2 Fluorescence microplate reader: Ex = 450 nm, Em = 550 nm for recombinant fluorescence titration and competition assays.
5.1.3 NanoBRET-compatible microplate reader: Donor emission = 450 nm, acceptor emission = 550 nm.
5.1.4 Native PAGE system with in-gel fluorescence and luminescence imaging.
5.2 Result analysis:
5.2.1 Fluorescence intensity changes: Dose-dependent increase in fluorescence upon LumiPK binding to recombinant PKL; dose-dependent decrease in fluorescence when LumiPK is displaced by competing allosteric modulators; rapid increase in intracellular fluorescence reaching a plateau within 4 min of LumiPK addition to live cells.
5.2.2 Fluorescence localization: Intracellular fluorescence in HEK293 cells, with stable signal over 60 min; in-gel fluorescence localized exclusively to tetrameric PKL (PKLwt and PKLNluc), no binding to monomeric/dimeric PKL or PKM2.
5.2.3 NanoBRET signal changes: Dose-dependent increase in BRET ratio with increasing LumiPK concentration in PKLNluc-transfected cells; dose-dependent decrease in BRET ratio when LumiPK is displaced by competing allosteric modulators.