Beta-glucuronidase protocols

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(Method)
(Assay 2)
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===Materials===
===Materials===
*1M Na<sub>2</sub>CO<sub>3</sub>
*1M Na<sub>2</sub>CO<sub>3</sub>
-
*100mM NaH<sub>2</sub>PO<sub>4</sub>
+
*100mM Sodium Phosphate buffer (pH=7)
 +
:*50mM NaH<sub>2</sub>PO<sub>4</sub>
 +
:*100mM Na<sub>2</sub>HPO<sub>4</sub>
*100mM EDTA
*100mM EDTA
*0.1% SDS (w/v in H<sub>2</sub>O)
*0.1% SDS (w/v in H<sub>2</sub>O)
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*Triton X-100
*Triton X-100
===Method===
===Method===
-
:1. Prepare 1500μL of GUS Buffer (for each sample to be measured) by adding:
+
:1. Grow culture until OD<sub>600</sub> is between 0.6 and 1.0
-
::*750μL of 100mM NaH<sub>2</sub>PO<sub>4</sub>
+
:2. DPrepare 10mL of GUS Buffer (measures 10 samples) by adding:
 +
::*3mL of 100mM Na<sub>2</sub>HPO<sub>4</sub>
 +
::*2mL of 100mM NaH<sub>2</sub>PO<sub>4</sub>
::*15μL 100mM EDTA
::*15μL 100mM EDTA
::*1.5μL Triton X-100
::*1.5μL Triton X-100

Revision as of 02:42, 26 January 2012

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 (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

  • 1M Na2CO3
  • 100mM Sodium Phosphate buffer (pH=7)
  • 50mM NaH2PO4
  • 100mM Na2HPO4
  • 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. Grow culture until OD600 is between 0.6 and 1.0
2. DPrepare 10mL of GUS Buffer (measures 10 samples) by adding:
  • 3mL of 100mM Na2HPO4
  • 2mL of 100mM NaH2PO4
  • 15μL 100mM EDTA
  • 1.5μL Triton X-100
  • 1μL β-mercaptoethanol
  • 720μL H2O
3. Measure and record the OD600 of the cell culture.
4. Pellet 1.5 ml of overnight culture by centrifugation for 1 minute.
5. Resuspend in 1ml gus buffer.
6. Pellet again by centrifugation.
7. Resuspend in 450μL GUS buffer.
8. Add 25ul chloroform and 12.5μL 0.1% SDS.
9. Vortex to mix for 10 secs.
10. Incubate for 10 min in 37°C water bath.
11. Add 50ul of 4-NPG solution and start the timer.
12. Incubate for 30 minutes in 37°C water bath.
13. After 30 minutes add 250μL 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.

Staining

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