Protein Overexpression and Extraction: Difference between revisions
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# Prepare and sterilize a minimal media where the carbon and nitrogen source can be controlled (M9 media). Make sure the volume of the media does not exceed half of the total volume of the flask that holds it (ex. 1L of media in a 2L flask). | # Prepare and sterilize a minimal media where the carbon and nitrogen source can be controlled (M9 media). Make sure the volume of the media does not exceed half of the total volume of the flask that holds it (ex. 1L of media in a 2L flask). | ||
# Add the appropriate drug to your media to select for your bacteria and plasmid. | # Add the appropriate drug to your media to select for your bacteria and plasmid. | ||
## 100mg/L ampicillin | |||
## 30mg/L kanamycin | |||
# Inoculate the media with your bacteria. | # Inoculate the media with your bacteria. | ||
# Allow the culture to grow to an A600 of 0.7-0.8. | # Allow the culture to grow to an A600 of 0.7-0.8. | ||
# Induce protein overexpression by adding IPTG to a final concentration of 0.4-2mM. Note the time. | # Induce protein overexpression by adding IPTG to a final concentration of 0.4-2mM. Note the time. | ||
## Start with 0.4mM (~0.1g/L). High concentrations of IPTG may be toxic. | |||
# Protein production should be monitored over 5 hours. At 1 hour time intervals, take a small amount of culture and save for SDS-PAGE analysis. | # Protein production should be monitored over 5 hours. At 1 hour time intervals, take a small amount of culture and save for SDS-PAGE analysis. | ||
## Future cultures can be harvested at the time when most of the target protein is present. | |||
# To harvest, spin the cultures down in the refrigerated centrifuge. | # To harvest, spin the cultures down in the refrigerated centrifuge. | ||
## I’ve found that 20 min at 4,000 rpm is sufficient. | |||
# Resuspend the bacteria in no more than 25 mL of lysis buffer. | # Resuspend the bacteria in no more than 25 mL of lysis buffer. | ||
Optional (but recommended): Add a protease inhibitor cocktail or a spatula tip of phenylmethanesulfonyl fluoride. | Optional (but recommended): Add a protease inhibitor cocktail or a spatula tip of phenylmethanesulfonyl fluoride. | ||
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Optional: Add 1mg of lysozyme per gram of cell pellet. Rock at RT for 20-30 minutes. | Optional: Add 1mg of lysozyme per gram of cell pellet. Rock at RT for 20-30 minutes. | ||
# Sonicate until the cell suspension changes color. It usually clears up or becomes a milky color. | # Sonicate until the cell suspension changes color. It usually clears up or becomes a milky color. | ||
## 10 bursts (10 sec) on a high power setting. | |||
# Spin down the cell debris. | # Spin down the cell debris. | ||
## 20 min at 4,000 rpm. | |||
12. The supernatant contains protein. Save the pellet in case your protein is stuck in an inclusion body. | 12. The supernatant contains protein. Save the pellet in case your protein is stuck in an inclusion body. | ||
Revision as of 20:21, 14 March 2017
Protein Overexpression and Extraction
- Prepare and sterilize a minimal media where the carbon and nitrogen source can be controlled (M9 media). Make sure the volume of the media does not exceed half of the total volume of the flask that holds it (ex. 1L of media in a 2L flask).
- Add the appropriate drug to your media to select for your bacteria and plasmid.
## 100mg/L ampicillin ## 30mg/L kanamycin
- Inoculate the media with your bacteria.
- Allow the culture to grow to an A600 of 0.7-0.8.
- Induce protein overexpression by adding IPTG to a final concentration of 0.4-2mM. Note the time.
## Start with 0.4mM (~0.1g/L). High concentrations of IPTG may be toxic.
- Protein production should be monitored over 5 hours. At 1 hour time intervals, take a small amount of culture and save for SDS-PAGE analysis.
- Future cultures can be harvested at the time when most of the target protein is present.
- To harvest, spin the cultures down in the refrigerated centrifuge.
## I’ve found that 20 min at 4,000 rpm is sufficient.
- Resuspend the bacteria in no more than 25 mL of lysis buffer.
Optional (but recommended): Add a protease inhibitor cocktail or a spatula tip of phenylmethanesulfonyl fluoride. Optional (but recommended): Freeze/flash freeze the resuspended cells and keep them in the freezer overnight. This helps to lyse the cells.
- Allow the cells to thaw and bring to room temperature.
Optional: Add 1mg of lysozyme per gram of cell pellet. Rock at RT for 20-30 minutes.
- Sonicate until the cell suspension changes color. It usually clears up or becomes a milky color.
## 10 bursts (10 sec) on a high power setting.
- Spin down the cell debris.
## 20 min at 4,000 rpm.
12. The supernatant contains protein. Save the pellet in case your protein is stuck in an inclusion body.