Protein Overexpression and Extraction - Revision history

Mjeppesen at 20:36, 21 July 2022

2022-07-21T20:36:14Z

← Older revision		Revision as of 20:36, 21 July 2022
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	# The supernatant contains protein. Save the pellet in case your protein is stuck in an inclusion body.		# The supernatant contains protein. Save the pellet in case your protein is stuck in an inclusion body.

	~~[[category:Protocols]]~~
	[[category:Protein_Preparation]]		[[category:Protein_Preparation]]
	[[category:Cell_Culturing]]		[[category:Cell_Culturing]]

Mjeppesen at 21:38, 13 November 2021

2021-11-13T21:38:51Z

← Older revision		Revision as of 21:38, 13 November 2021
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	Protein Overexpression and Extraction		Protein Overexpression and Extraction
	See also [[Protein_expression \| Protein Expression]]		- See also [[Protein_expression \| Protein Expression]]

	# 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).

Mjeppesen at 21:38, 13 November 2021

2021-11-13T21:38:38Z

← Older revision		Revision as of 21:38, 13 November 2021
Line 1:		Line 1:
	Protein Overexpression and Extraction		Protein Overexpression and Extraction
			See also [[Protein_expression \| Protein Expression]]

	# 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).

Mjeppesen: LearningSorting

2021-09-11T04:57:32Z

LearningSorting

← Older revision		Revision as of 04:57, 11 September 2021
Line 24:		Line 24:
	# The supernatant contains protein. Save the pellet in case your protein is stuck in an inclusion body.		# The supernatant contains protein. Save the pellet in case your protein is stuck in an inclusion body.

	[[category:~~Protocols\|~~Protocols]]		[[category:Protocols]]
	[[category:Protein_Preparation]]		[[category:Protein_Preparation]]
	[[category:Cell_Culturing]]		[[category:Cell_Culturing]]

Mjeppesen: Added to Category

2021-09-11T03:11:08Z

Added to Category

← Older revision		Revision as of 03:11, 11 September 2021
Line 23:		Line 23:
	## 20 min at 4,000 rpm.		## 20 min at 4,000 rpm.
	# The supernatant contains protein. Save the pellet in case your protein is stuck in an inclusion body.		# The supernatant contains protein. Save the pellet in case your protein is stuck in an inclusion body.

			[[category:Protocols\|Protocols]]
			[[category:Protein_Preparation]]
			[[category:Cell_Culturing]]

Jcatazaro at 20:23, 14 March 2017

2017-03-14T20:23:23Z

← Older revision		Revision as of 20:23, 14 March 2017
Line 22:		Line 22:
	# Spin down the cell debris.		# Spin down the cell debris.
	## 20 min at 4,000 rpm.		## 20 min at 4,000 rpm.
			# 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.

Jcatazaro at 20:22, 14 March 2017

2017-03-14T20:22:42Z

Jcatazaro at 20:21, 14 March 2017

2017-03-14T20:21:04Z

Jcatazaro at 20:19, 14 March 2017

2017-03-14T20:19:25Z

← Older revision		Revision as of 20:19, 14 March 2017
Line 10:		Line 10:
	* Start with 0.4mM (~0.1g/L). High concentrations of IPTG may be toxic.		* 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.		** 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.		* I’ve found that 20 min at 4,000 rpm is sufficient.

Jcatazaro: Created page with "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 m..."

2017-03-14T20:18:46Z

Created page with "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 m..."

New page

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.

← Older revision		Revision as of 20:22, 14 March 2017
Line 3:		Line 3:
	# 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		## 100mg/L ampicillin
	## 30mg/L kanamycin		## 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.		## 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.		## 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.		## 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.
	Optional (but recommended): Freeze/flash freeze the resuspended cells and keep them in the freezer overnight. This helps to lyse the cells.		##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.		# 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.		##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.		## 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.		## 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.

← Older revision		Revision as of 20:21, 14 March 2017
Line 3:		Line 3:
	# 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		## 100mg/L ampicillin
	* 30mg/L kanamycin		## 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.		## 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.		## 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.		## 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.
Line 19:		Line 19:
	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.		## 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.		## 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.