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.

Plate Screen

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.

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

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.

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 (200mM 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 20μL of 4-NPG stock solution.
8. Let the reaction run for 10-30 minutes.
9. Add 200μL Stop Solution to halt the reaction.
10. Centrifuge to pellet cell debris.
11. Measure the OD₄₀₅ of the stopped reaction.

Notes

• Use this one for E. coli.

Assay 2

Materials

• 100mM sodium phosphate buffer (pH=7)

• 40mM NaH₂PO₄
• 60mM Na₂HPO₄

• 0.1M potassium chloride solution
• 10mM magnesium sulfate solution
• 1M Na₂CO₃
• 4-Nitrophenyl β-D-glucuronide (4-NPG) stock solution (10mg/ml in 50mM sodium phosphate buffer (pH=7)) only make 1mL of this!!!
• β-mercaptoethanol
• 10% Triton X-100 (in water)

Method

1. Grow culture until OD₆₀₀ is between 0.6 and 1.0

2. Prepare 10mL of GUS Buffer (measures 10 samples) by adding:

• 5mL of sodium phosphate buffer (pH=7)
• 3mL H₂O
• 1mL of potassium chloride solution
• 1mL of magnesium sulfate solution
• 35μL β-mercaptoethanol
• 20mg Lysozyme

3. Pellet 1.5 ml of culture by centrifugation for 1 minute.

4. Resuspend in 1ml 100mM sodium phosphate buffer.

5. Pellet again by centrifugation.

6. Resuspend in 750μL GUS buffer.

7. Vortex briefly to mix.

8. Incubate for 30 min in 37°C water bath.

9. Add 8ul of 10% Triton-X.

10. Vortex briefly and incubate on ice for 5 mins.

11. Add 80ul of 4-NPG solution and start the timer.

12. Incubate in 37°C water bath.

13. When the color is clearly yellow (between 10 and 30 mins), stop reaction by adding 300μL 1M Na₂CO₃

14. Record the time.

15. Centrifuge the reaction for 1 minute at full speed.

16. Measure the OD₄₀₅ of the supernatant.

Notes

Use this one for Lactobacillus spp.