Beta-glucuronidase protocols

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===Materials===
===Materials===
*GUS Buffer (50mM NaH<sub>2</sub>PO<sub>4</sub>, 10mM β-mercaptoethanol, 1mM EDTA and 0.1% Triton X-100)
*GUS Buffer (50mM NaH<sub>2</sub>PO<sub>4</sub>, 10mM β-mercaptoethanol, 1mM EDTA and 0.1% Triton X-100)
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===Method===
===Method===

Revision as of 02:07, 5 July 2011

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)
  • Solubility 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 Permeability Solution to cell suspension.
  4. Incubate at 37°C for 30-60 minutes.
  5. Add 5μL of X-gluc stock solution.
  6. Color should develop shortly.

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

Materials

  • GUS Buffer (50mM NaH2PO4, 10mM β-mercaptoethanol, 1mM EDTA and 0.1% Triton X-100)

Method

Notes

Staining

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