In vitro GCase activity assay (total cell lysate)
Federico Bertoli, Michela Deleidi
Abstract
Glucocerebrosidase is a lysosomal enzyme that catalyzes the hydrolysis of glucosylceramide (GlcCer), a membrane glyco-sphingolipid, to ceramide and glucose. This assay detects GBA activity by using a fluorogenic substrate that reacts with cell lysates previously treated with or without CBE (GBA1 inhibitor).
Attachments
Steps
Sample Lysis
Suspend samples in 50µL of 1% Triton extraction buffer.
Homogenize with a Dounce homogenizer for 25 strokes.
Rotate samples for 0h 30m 0s at 4°C.
Centrifuge at 13500x g,0h 0m 0s, 4°C for 0h 15m 0s.
Collect supernatants.
Substrate preparation
Add 20.30mg 4-Methylumbelliferyl-β-D-glucopyranoside for 10mL ddH2O of substrate (6millimolar (mM)).
Incubate at 55°C and vortex every 0h 5m 0s until dissolved (approx. 0h 30m 0s).
Store at 4°C until needed.
Sample preparation
Add the equivalent of 10µg total protein in ddH2O to reach a final 45µL volume.
Add to each 25µL McIlave Buffer 6 and mix it.
Divide the overall 70µL volume into two tubes (35µL each).
Incubate one tube with 5µL CBE 1millimolar (mM) at 4Room temperature for 0h 30m 0s .
Incubate the other one with 5µL ddH2O at 4Room temperature for 0h 30m 0s.
Enzymatic reaction
Add 25µL substrate to each reaction tube.
Incubate at 37°C for 2h 0m 0s.
Measurement
Take 10µL of each reaction tube into a 96-well plate (in triplicate).
Add 90µL 0.2Molarity (M) glycine 10.2 to each well to stop the reaction.
Measure fluorescence: Excitation 355nm, Emission 460nm.
