Beta-glucuronidase protocols: Difference between revisions
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==Introduction== | ==Introduction== | ||
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*Media of choice | *Media of choice | ||
*Agar | *Agar | ||
*X-gluc stock solution (50mg/mL) | *X-gluc stock solution (50mg/mL in DMF) | ||
===Method=== | ===Method=== | ||
#Prepare your liquid media and add the desired amount of agar (usually 1-2%). | #Prepare your liquid media and add the desired amount of agar (usually 1-2%). | ||
| Line 15: | Line 13: | ||
#If desired, add antibiotic. | #If desired, add antibiotic. | ||
#Pour plates. | #Pour plates. | ||
===Notes=== | |||
==Culture Screen== | |||
===Materials=== | |||
*Cultured Cells | |||
*Suspension Buffer (50mM NaH<sub>2</sub>PO<sub>4</sub> | |||
*X-gluc stock solution (50mg/mL) | |||
*Solubility 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 Permeability Solution to cell suspension. | |||
#Incubate at 37°C for 30-60 minutes. | |||
#Add 5μL of X-gluc stock solution. | |||
#Color should develop shortly. | |||
===Notes=== | ===Notes=== | ||
==Assay== | ==Assay== | ||
===Materials=== | ===Materials=== | ||
*GUS Buffer (50mM NaH<sub>2</sub>PO<sub>4</sub>, 10mM β-mercaptoethanol, 1mM EDTA and 0.1% Triton X-100) | |||
===Method=== | ===Method=== | ||
Revision as of 23:01, 4 July 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)
- Solubility 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 Permeability Solution to cell suspension.
- Incubate at 37°C for 30-60 minutes.
- Add 5μL of X-gluc stock solution.
- Color should develop shortly.
Notes
Assay
Materials
- GUS Buffer (50mM NaH2PO4, 10mM β-mercaptoethanol, 1mM EDTA and 0.1% Triton X-100)
