Beta-glucuronidase protocols

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Contents

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.

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

  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 (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.

Notes

Assay 1

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

  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.

Notes

Staining

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