Beta-glucuronidase protocols

Introduction
These are methods to screen for and assay the activity of the common reporter enzyme β-Glucuronidase (GUS) activity. Because the catalytic activity of β-Glucuronidase is very similar to β-Galactosidase (LacZ) these protocols are also very similar to the LacZ protocols.

Materials

 * Media of choice
 * Agar
 * X-gluc stock solution (50mg/mL in DMF)

Method

 * 1) Prepare your liquid media and add the desired amount of agar (usually 1-2%).
 * 2) Autoclave the media for the requisite time.
 * 3) Add 1.2μL of X-gluc stock solution for every mL of media (e.g. if making 1L of media add 1.2ml of stock solution)
 * 4) If desired, add antibiotic.
 * 5) Pour plates.

Materials

 * Cultured Cells
 * Suspension Buffer (50mM NaH2PO4
 * X-gluc stock solution (50mg/mL)
 * Premeabilization Solution (9:1 acetone to toluene (v/v))

Method

 * 1) Pellet 1ml of culture by centrifugation
 * 2) Discard the supernatant and resuspend the pellet in 400μL Suspension Buffer
 * 3) Add 25ul of Permeabilization Solution to cell suspension.
 * 4) Incubate at 37°C for 30-60 minutes.
 * 5) Add 5μL of X-gluc stock solution.
 * 6) A green/blue color should develop shortly in positive cultures.

Materials

 * Suspension Solution (50mM NaH2PO4)
 * Permeabilization solution (9:1 acetone to toluene (v/v))
 * GUS Buffer (50mM NaH2PO4, 10mM β-mercaptoethanol, 1mM EDTA and 0.1% Triton X-100)
 * 4-Nitrophenyl β-D-glucuronide (4-NPG) stock solution (10mg/ml in 50mM NaH2PO4)
 * Stop Buffer (1M Na2CO3)

Method

 * 1) Measure and record the OD600 of the cell culture
 * 2) Pellet 1mL of culture by centrifugation.
 * 3) Resuspend the pellet in 400μL of Suspension Solution.
 * 4) Add 25ul of permeabilization solution.
 * 5) Incubate for 30 to 60 minutes at 37°C
 * 6) Take 50μL of this cell suspension and add it to 200μL of GUS Buffer.
 * 7) Add 12.5μL of 4-NPG stock solution.
 * 8) Let the reaction run for 10-30 minutes.
 * 9) Add 260μL Stop Solution to halt the reaction.
 * 10) Measure the OD405 of the stopped reaction.

Materials

 * Permeabilization solution (100mM NaH2PO4, 20mM KCl, 2mM MgSO4, 0.8 mg/mL CTAB, 0.4 mg/mL sodium deoxycholate, 5.4 μL/mL β-mercaptoethanol)
 * Substrate Solution (50mM NaH2PO4, 10mM β-mercaptoethanol, 1mM EDTA and 0.1% Triton X-100, 1mg/ml 4-NPG)
 * Stop Buffer (1M Na2CO3)

Method

 * 1) Measure and record the OD600 of the cell culture
 * 2) Add 20μL of cell culture to 80μL of Permeabilization Solution (this will halt protein production).
 * 3) Incubate for 20 to 30 minutes at 37°C to permeabilize cells.
 * 4) Add 600ul of Substrate Solution.
 * 5) Let the reaction run for 10-30 minutes (be sure to record the run time).
 * 6) Add 700μL Stop Solution to halt the reaction.
 * 7) Centrifuge the reaction for 5 minutes at full speed.
 * 8) Measure the OD405 of the supernatant.