RBM1-151

RBM1-151 (20 μM, 1 h) is hydrolyzed to generate RBM1-151-NH₂ only by acid ceramidase (AC) in cell-free systems (using A375/AC cell lysates)^[1].
RBM1-151 (0-20 μM , 30 min) is hydrolyzed by A375/AC cell lysates (20 μg) in acid buffer, with kinetic parameters ^appK_m = 7.0 μM and ^appV_max = 99.3 nM/min calculated via Michaelis-Menten analysis^[1].
RBM1-151 (5 μM, 3 h) shows higher hydrolysis (fluorescent product) in HEK293/NAAA cell lysates (10 μg, overexpressing NAAA) than HEK293/mock lysates^[1].
RBM1-151 (20 μM, 3 h) produces minimal fluorescence in Farber disease (FD) cells (lacking AC), while significantly higher fluorescence in FD/AC cells (overexpressing AC) in intact cells^[1].
RBM1-151 (20 μM, 3 h) shows higher hydrolysis in A375/AC cells (overexpressing AC) than A375/WT cells^[1].
Guide (The following is our recommended protocol. This protocol is only a guide and should be modified according to your specific needs).
1. Cell preparation
1.1 Suspended cells (e.g., AML cell lines: MM-6, HL-60): Culture cells in RPMI-1640 medium supplemented with 20% FBS; adjust cell density to 2 × 10⁴ cells per well before plating.
1.2 Adherent cells (e.g., melanoma cell lines: A375/AC, C8161; HEK293): Culture cells in Dulbecco's modified Eagle's medium (high glucose) supplemented with 10% FBS and 1% penicillin/streptomycin; plate 2 × 10⁴ cells per well in 96-well plates and incubate overnight to allow adherence.
Note: For cell lines requiring antibiotic selection (e.g., A375/AC with blasticidin and hygromycin), remove antibiotics from the medium before the assay to avoid interference.
2. RBM1-151 incubation and signal detection
2.1 Add 50 μL of 20 μM RBM1-151 working solution (in 20% FBS medium) to each well (final RBM1-151 concentration: 20 μM).
2.2 Incubate the plate at 37°C with 5% CO2 for 1 hour.
2.3 Stop the reaction by adding 25 μL of 100% methanol per well.
2.4 Immediately add 100 μL of NaIO4 solution (2.5 mg/mL in 100 mM glycine-NaOH buffer pH 10.6) to each well.
2.5 Incubate the plate at 37°C with 5% CO2 for 30 minutes in the dark.
2.6 Measure fluorescence using a microtiter plate reader; subtract the background signal from wells containing medium and RBM1-151 but no cells.

MedChemExpress (MCE) has not independently confirmed the accuracy of these methods. They are for reference only.