Beta-glucuronidase protocols: Difference between revisions
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:1. Prepare 1500μL of GUS solution (for each sample to be measured) by adding: | :1. Prepare 1500μL of GUS solution (for each sample to be measured) by adding: | ||
::*750μL of 100mM NaH<sub>2</sub>PO<sub>4</sub> | ::*750μL of 100mM NaH<sub>2</sub>PO<sub>4</sub> | ||
::*15μL 100mM EDTA | ::*15μL 100mM EDTA | ||
::* | ::*50ul 0.1% SDS | ||
::*1.5μL Triton X-100 | ::*1.5μL Triton X-100 | ||
::*1μL β-mercaptoethanol | ::*1μL β-mercaptoethanol | ||
Line 74: | Line 73: | ||
:3. Measure and record the OD<sub>600</sub> of the cell culture.<br> | :3. Measure and record the OD<sub>600</sub> of the cell culture.<br> | ||
:4. Add 50μL of cell culture each tube.<br> | :4. Add 50μL of cell culture each tube.<br> | ||
:5. Incubate for | :5. Add 25ul chloroform and vortex to mix. | ||
: | :6. Incubate for 10 min at 37°C.<br> | ||
: | :7. Add 50ul of 4-NPG solution and start the timer.<br> | ||
: | :8. At 10, 20, and 30 minutes add 400μL 1M Na<sub>2</sub>CO<sub>3</sub> to stop the reaction.<br> | ||
: | :9. Centrifuge the reaction for 1 minute at full speed.<br> | ||
:10. Measure the OD<sub>405</sub> of the supernatant.<br> | |||
===Notes=== | ===Notes=== |
Revision as of 10:42, 12 August 2011
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 13μL 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
- Use this one for E. coli.
Assay 2
Materials
- 1M Na2CO3
- 100mM NaH2PO4
- 100mM EDTA
- 0.1% SDS (w/v in H2O)
- 4-Nitrophenyl β-D-glucuronide (4-NPG) stock solution (10mg/ml in 50mM NaH2PO4)
- β-mercaptoethanol
- Chloroform
- Triton X-100
Method
- 1. Prepare 1500μL of GUS solution (for each sample to be measured) by adding:
- 750μL of 100mM NaH2PO4
- 15μL 100mM EDTA
- 50ul 0.1% SDS
- 1.5μL Triton X-100
- 1μL β-mercaptoethanol
- 700μL H2O
- 2. Aliquot into tubes containing 500μL each.
- 3. Measure and record the OD600 of the cell culture.
- 4. Add 50μL of cell culture each tube.
- 5. Add 25ul chloroform and vortex to mix.
- 6. Incubate for 10 min at 37°C.
- 7. Add 50ul of 4-NPG solution and start the timer.
- 8. At 10, 20, and 30 minutes add 400μL 1M Na2CO3 to stop the reaction.
- 9. Centrifuge the reaction for 1 minute at full speed.
- 10. Measure the OD405 of the supernatant.
Notes
Use this one for Lactobacillus spp.