Beta-glucuronidase protocols: Difference between revisions
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*Suspension Buffer (50mM NaH<sub>2</sub>PO<sub>4</sub> | *Suspension Buffer (50mM NaH<sub>2</sub>PO<sub>4</sub> | ||
*X-gluc stock solution (50mg/mL) | *X-gluc stock solution (50mg/mL) | ||
* | *Premeabilization Solution (9:1 acetone to toluene (v/v)) | ||
===Method=== | ===Method=== | ||
#Pellet 1ml of culture by centrifugation | #Pellet 1ml of culture by centrifugation | ||
#Discard the supernatant and resuspend the pellet in 400μL Suspension Buffer | #Discard the supernatant and resuspend the pellet in 400μL Suspension Buffer | ||
#Add 25ul of | #Add 25ul of Permeabilization Solution to cell suspension. | ||
#Incubate at 37°C for 30-60 minutes. | #Incubate at 37°C for 30-60 minutes. | ||
#Add 5μL of X-gluc stock solution. | #Add 5μL of X-gluc stock solution. | ||
#A green/blue color should develop shortly in positive cultures. | #A green/blue color should develop shortly in positive cultures. | ||
===Notes=== | ===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. | *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== | ==Assay 1== | ||
===Materials=== | ===Materials=== | ||
*Suspension Solution (50mM NaH<sub>2</sub>PO<sub>4</sub>) | |||
*Permeabilization solution (9:1 acetone to toluene (v/v)) | |||
*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) | ||
*4-Nitrophenyl β-D-glucuronide (4-NPG) stock solution (10mg/ml in 50mM NaH<sub>2</sub>PO<sub>4</sub>) | |||
*Stop Buffer (1M Na<sub>2</sub>CO<sub>3</sub>) | |||
===Method=== | ===Method=== | ||
#Measure and record the OD<sub>600</sub> 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 12.5μL of 4-NPG stock solution. | |||
#Let the reaction run for 10-30 minutes. | |||
#Add 260μL Stop Solution to halt the reaction. | |||
#Measure the OD<sub>405</sub> of the stopped reaction. | |||
===Notes=== | ===Notes=== | ||
==Assay 1== | |||
===Materials=== | |||
*Suspension Solution (50mM NaH<sub>2</sub>PO<sub>4</sub>) | |||
*Permeabilization solution (100mM NaH<sub>2</sub>PO<sub>4</sub>, 20mM KCl, 2mM MgSO4, 0.8 mg/mL CTAB, 0.4 mg/mL sodium deoxycholate, 5.4 μL/mL β-mercaptoethanol) | |||
*Substrate Solution (50mM NaH<sub>2</sub>PO<sub>4</sub>, 10mM β-mercaptoethanol, 1mM EDTA and 0.1% Triton X-100, 1mg/ml 4-NPG) | |||
*Stop Buffer (1M Na<sub>2</sub>CO<sub>3</sub>) | |||
===Method=== | |||
#Measure and record the OD<sub>600</sub> of the cell culture | |||
#Add 20μL of cell culture to 80μL of Permeabilization Solution (this will halt protein production). | |||
#Incubate for 20 to 30 minutes at 37°C to permeabilize cells. | |||
#Add 600ul of Substrate Solution. | |||
#Let the reaction run for 10-30 minutes (be sure to record the run time). | |||
#Add 700μL Stop Solution to halt the reaction. | |||
#Centrifuge the reaction for 5 minutes at full speed. | |||
#Measure the OD<sub>405</sub> of the supernatant. | |||
===Notes=== | |||
==Staining== | ==Staining== | ||
Revision as of 00:05, 5 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)
- 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 12.5μL 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
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
- Measure and record the OD600 of the cell culture
- Add 20μL of cell culture to 80μL of Permeabilization Solution (this will halt protein production).
- Incubate for 20 to 30 minutes at 37°C to permeabilize cells.
- Add 600ul of Substrate Solution.
- Let the reaction run for 10-30 minutes (be sure to record the run time).
- Add 700μL Stop Solution to halt the reaction.
- Centrifuge the reaction for 5 minutes at full speed.
- Measure the OD405 of the supernatant.