Beta-glucuronidase protocols
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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.
Plate Screen
Materials
- Media of choice
- Agar
- X-gluc stock solution (50mg/mL in DMF)
Method
- Prepare your liquid media and add the desired amount of agar (usually 1-2%).
- Autoclave the media for the requisite time.
- 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)
- If desired, add antibiotic.
- Pour plates.
Notes
Culture Screen
Materials
- Cultured Cells
- Suspension Buffer (50mM NaH2PO4
- X-gluc stock solution (50mg/mL)
- Premeabilization Solution (9:1 acetone to toluene (v/v))
Method
- Pellet 1ml of culture by centrifugation
- Discard the supernatant and resuspend the pellet in 400μL Suspension Buffer
- Add 25ul of Permeabilization Solution to cell suspension.
- Incubate at 37°C for 30-60 minutes.
- Add 5μL of X-gluc stock solution.
- A green/blue color should develop shortly in positive cultures.
Notes
- Depending on what cells you're using, the solvent mix may not permeabilize your cells. In such a case you might attempt using 25μL of chloroform or 12.5μL of 1%SDS or both.
Assay 1
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
- Measure and record the OD600 of the cell culture
- Pellet 1mL of culture by centrifugation.
- Resuspend the pellet in 400μL of Suspension Solution.
- Add 25ul of permeabilization solution.
- Incubate for 30 to 60 minutes at 37°C
- Take 50μL of this cell suspension and add it to 200μL of GUS Buffer.
- Add 12.5μL of 4-NPG stock solution.
- Let the reaction run for 10-30 minutes.
- Add 260μL Stop Solution to halt the reaction.
- Measure the OD405 of the stopped reaction.
Notes
Assay 2
Materials
- Suspension Solution (50mM NaH2PO4)
- 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
- Measure and record the OD600 of the cell culture
- Add 20μL of cell culture to 80μL of Permeabilization Solution (this will halt protein production).
- Incubate for 20 to 30 minutes at 37°C to permeabilize cells.
- Add 600ul of Substrate Solution.
- Let the reaction run for 10-30 minutes (be sure to record the run time).
- Add 700μL Stop Solution to halt the reaction.
- Centrifuge the reaction for 5 minutes at full speed.
- Measure the OD405 of the supernatant.