Western Blotting - Revision history

Davebrid: /* Materials */ Added product numbers for some materials

2025-01-20T17:46:54Z

Materials: Added product numbers for some materials

← Older revision		Revision as of 17:46, 20 January 2025
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	==Materials==		==Materials==
			*Protein Markers, we use SeaBlue Plus2 from Invitrogen (cat #LC5925)
			*Nitrocellulose, we use BioRad 0.45 um membrane from a roll (cat #1620115) and cut to the size of a gel
			*Filter Paper, we use BioRad extra thick paper (cat 1703966)
	*Transfer Buffer (200 mL Methanol, 100 mL 10X [[ Transfer Buffer ]] to final 1L volume)		*Transfer Buffer (200 mL Methanol, 100 mL 10X [[ Transfer Buffer ]] to final 1L volume)
	*Transfer Apparatus, either Bio-Rad or Invitrogen		*Transfer Apparatus, either Bio-Rad or Invitrogen

Davebrid: Added link to video

2025-01-20T16:39:17Z

Added link to video

← Older revision		Revision as of 16:39, 20 January 2025
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	#Turn on heat block to 85 degrees		#Turn on heat block to 85 degrees
	#Run SDS-PAGE gel using [[ SDS-PAGE Running Buffer ]] and prepare diluted transfer buffer		#Run SDS-PAGE gel using [[ SDS-PAGE Running Buffer ]] and prepare diluted transfer buffer
	## Use a prepared ~~4-12% tris~~ gel (in the 4 degree). Remove it from the packaging, remove the white strip of tape from the bottom back, and gently pull the comb out and rinse with water.		## Use a prepared gel (in the 4 degree). Remove it from the packaging, remove the white strip of tape from the bottom back, and gently pull the comb out and rinse with water. Pick a percent gel based on your expected protein size.
	##Load into gel tank. Fill with SDS page Running buffer (1X) to the fill line in the front and halfway up the back		##Load into gel tank. Fill with SDS page Running buffer (1X) to the fill line in the front and halfway up the back. For a video on how to use these gel tanks see https://youtu.be/aedIjTtNEus
	##Boil sample at 85 degrees for ~3 min		##Boil sample at 85 degrees for ~3 min
	##Load 3 microliters of protein ladder (purple top) (in the 4 degree), and 10 microliters of each sample into separate wells.		##Load 3 microliters of protein ladder (purple top) (in the 4 degree), and 10 microliters of each sample into separate wells.

Davebrid: /* SOP */ Added links to SOPs

2025-01-20T16:31:55Z

SOP: Added links to SOPs

← Older revision		Revision as of 16:31, 20 January 2025
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	==SOP==		==SOP==
	*~~Irritant~~		* [[SOP_-_Irritants\|SOP - Irritants]]
	*Electrophoresis		* [[SOP - Electrophoresis]]

	==Materials==		==Materials==

Reddj at 15:58, 26 March 2021

2021-03-26T15:58:22Z

← Older revision		Revision as of 15:58, 26 March 2021
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	#Wash blot every 5 minutes for 15 min with TBST.		#Wash blot every 5 minutes for 15 min with TBST.
	#Rinse once or twice with double distilled water.		#Rinse once or twice with double distilled water.
	#Scan dry blot using LiCor Odyssey.		#Scan dry blot using the LiCor Odyssey [[Scanning and Analyzing Western Blots Using LiCor Odyssey]].

	==If Using LiCor==		==If Using LiCor==
	~~#Start -> New -> Scan Image -> Login -> Peloquin -> Password Located in Desk -> Select Dimensions -> Start Scan~~

			[[Scanning and Analyzing Western Blots Using LiCor Odyssey]]


	[[ Category: Western Blotting ]]		[[ Category: Western Blotting ]]

Reddj: removed ECL instructions- JR

2021-03-26T13:54:04Z

removed ECL instructions- JR

Reddj at 14:37, 8 January 2019

2019-01-08T14:37:40Z

← Older revision		Revision as of 14:37, 8 January 2019
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	==SOP==		==SOP==
	*Irritant, Electrophoresis		*Irritant
			*Electrophoresis

	==Materials==		==Materials==

Kkistler: Corrections to protocol

2019-01-07T20:20:43Z

Corrections to protocol

Kkistler: Added SOP

2019-01-07T20:16:27Z

Added SOP

← Older revision		Revision as of 20:16, 7 January 2019
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			==SOP==
			*Irritant, Electrophoresis

	==Materials==		==Materials==
	*Transfer Buffer (200 mL Methanol, 100 mL 10X [[ Transfer Buffer ]] to final 1L volume)		*Transfer Buffer (200 mL Methanol, 100 mL 10X [[ Transfer Buffer ]] to final 1L volume)

Mollyec: /* Protocol */

2019-01-07T18:07:27Z

Protocol

← Older revision		Revision as of 18:07, 7 January 2019
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	##Load 3 microliters of protein ladder (purple), and 10 microliters of each sample into separate wells.		##Load 3 microliters of protein ladder (purple), and 10 microliters of each sample into separate wells.
	## Place top on tank, plug into power source and run at 125 Volts until samples and ladder reach the bottom of the gel		## Place top on tank, plug into power source and run at 125 Volts until samples and ladder reach the bottom of the gel
	#Make sandwich (black side, sponge, filter paper, ~~Gel~~, ~~Nitrocellulose~~, ~~Filter~~ paper, ~~Sponge~~, clear side), ensuring no bubbles between layers with black piece on bottom and layer as above. Place in apparatus so that the black sandwich touches the black transfer piece. Fill with transfer buffer.		#Make sandwich (black side, sponge, filter paper, gel, nitrocellulose, filter paper, sponge, clear side), ensuring no bubbles between layers with black piece on bottom and layer as above. Place in apparatus so that the black sandwich touches the black transfer piece. Fill with transfer buffer.
	#Transfer 4h at 75V (in cold room) or overnight at 35V (room temp).		#Transfer 4h at 75V (in cold room) or overnight at 35V (room temp).
	#Stain for total protein with Revert total protein stain on rocker for 5 minutes --when finished pour total protein stain back in bottle for later use!		#Stain for total protein with Revert total protein stain on rocker for 5 minutes --when finished pour total protein stain back in bottle for later use!

Mollyec: /* Protocol */

2019-01-07T18:07:05Z

Protocol

← Older revision		Revision as of 18:07, 7 January 2019
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	##Load 3 microliters of protein ladder (purple), and 10 microliters of each sample into separate wells.		##Load 3 microliters of protein ladder (purple), and 10 microliters of each sample into separate wells.
	## Place top on tank, plug into power source and run at 125 Volts until samples and ladder reach the bottom of the gel		## Place top on tank, plug into power source and run at 125 Volts until samples and ladder reach the bottom of the gel
	#Make sandwich, ensuring no bubbles between layers with black piece on bottom and layer as above. Place in apparatus so that the black sandwich touches the black transfer piece. Fill with transfer buffer.		#Make sandwich (black side, sponge, filter paper, Gel, Nitrocellulose, Filter paper, Sponge, clear side), ensuring no bubbles between layers with black piece on bottom and layer as above. Place in apparatus so that the black sandwich touches the black transfer piece. Fill with transfer buffer.
	#Transfer 4h at 75V (in cold room) or overnight at 35V (room temp).		#Transfer 4h at 75V (in cold room) or overnight at 35V (room temp).
	#Stain for total protein with Revert total protein stain on rocker for 5 minutes --when finished pour total protein stain back in bottle for later use!		#Stain for total protein with Revert total protein stain on rocker for 5 minutes --when finished pour total protein stain back in bottle for later use!

← Older revision		Revision as of 13:54, 26 March 2021
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	##Load into gel tank. Fill with SDS page Running buffer (1X) to the fill line in the front and halfway up the back		##Load into gel tank. Fill with SDS page Running buffer (1X) to the fill line in the front and halfway up the back
	##Boil sample at 85 degrees for ~3 min		##Boil sample at 85 degrees for ~3 min
	##Load 3 microliters of protein ladder (purple) (in the 4 degree), and 10 microliters of each sample into separate wells.		##Load 3 microliters of protein ladder (purple top) (in the 4 degree), and 10 microliters of each sample into separate wells.
	## Place top on tank, plug into power source and run at 125 Volts until samples and ladder reach the bottom of the gel		## Place top on tank, plug into power source and run at 125 Volts until samples and ladder reach the bottom of the gel. (Tip: Gel runs more evenly if you start a lower V and increase once the samples have run down 1/3 of the gel.)
	#Make sandwich (black side, sponge, filter paper, gel, nitrocellulose, filter paper, sponge, clear side), ensuring no bubbles between layers with black piece on bottom and layer as above. Place in apparatus so that the black sandwich touches the black transfer piece. Fill with transfer buffer.		#Make sandwich (black side, sponge, filter paper, gel, nitrocellulose, filter paper, sponge, clear side), ensuring no bubbles between layers with black piece on bottom and layer as above. Place in apparatus so that the black sandwich touches the black transfer piece. Fill with transfer buffer.
	#Transfer 4h at 75V (in cold room) or overnight at 35V (room temp).		#Transfer 4h at 75V (in cold room) or overnight at 35V (room temp with an ice pack).
	#Stain for total protein with Revert total protein stain on rocker for 5 minutes --when finished pour total protein stain back in bottle for later use!		#Stain for total protein with Revert total protein stain on rocker for 5 minutes --when finished pour total protein stain back in bottle for later use!
	#Rinse twice in revert wash solution (60ml MeOH, 13.4 ml Acetic Acid, 126.6 ml Water)		#Rinse twice in revert wash solution (60ml MeOH, 13.4 ml Acetic Acid, 126.6 ml Water)
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	#Incubate with appropriate secondary antibody (10 000X) for 45min-1h (20 ml 2% BSA 1ul of both secondary antibodies, all found in fridge Ab stocks with blue dots on top)		#Incubate with appropriate secondary antibody (10 000X) for 45min-1h (20 ml 2% BSA 1ul of both secondary antibodies, all found in fridge Ab stocks with blue dots on top)
	#Wash blot every 5 minutes for 15 min with TBST.		#Wash blot every 5 minutes for 15 min with TBST.
	#Rinse once or twice with double distilled water		#Rinse once or twice with double distilled water.
	~~#Prepare ECL by mixing one volume of the black bottle solution with one volume of the white bottle solution (be careful not to cross-contaminate ECL bottles with wrong solution)~~.		#Scan dry blot using LiCor Odyssey.
	#~~Drain excess buffer from~~ blot ~~and cover with ECL for about a minute~~
	~~#Drain excess ECL from blot, cover with saran wrap and expose film~~

	==If Using LiCor==		==If Using LiCor==

← Older revision		Revision as of 20:20, 7 January 2019
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	==Protocol==		==Protocol==
			#Turn on heat block to 85 degrees
	#Run SDS-PAGE gel using [[ SDS-PAGE Running Buffer ]] and prepare diluted transfer buffer		#Run SDS-PAGE gel using [[ SDS-PAGE Running Buffer ]] and prepare diluted transfer buffer
	## Use a prepared 5-12% tris gel (in the 4 degree). Remove it from the packaging, remove the white strip of tape from the bottom back, and gently pull the comb out and rinse with water.		## Use a prepared 4-12% tris gel (in the 4 degree). Remove it from the packaging, remove the white strip of tape from the bottom back, and gently pull the comb out and rinse with water.
	##Load into gel tank. Fill with SDS page Running buffer (1X) to the fill line in the front and halfway up the back		##Load into gel tank. Fill with SDS page Running buffer (1X) to the fill line in the front and halfway up the back
	##Load 3 microliters of protein ladder (purple), and 10 microliters of each sample into separate wells.		##Boil sample at 85 degrees for ~3 min
			##Load 3 microliters of protein ladder (purple) (in the 4 degree), and 10 microliters of each sample into separate wells.
	## Place top on tank, plug into power source and run at 125 Volts until samples and ladder reach the bottom of the gel		## Place top on tank, plug into power source and run at 125 Volts until samples and ladder reach the bottom of the gel
	#Make sandwich (black side, sponge, filter paper, gel, nitrocellulose, filter paper, sponge, clear side), ensuring no bubbles between layers with black piece on bottom and layer as above. Place in apparatus so that the black sandwich touches the black transfer piece. Fill with transfer buffer.		#Make sandwich (black side, sponge, filter paper, gel, nitrocellulose, filter paper, sponge, clear side), ensuring no bubbles between layers with black piece on bottom and layer as above. Place in apparatus so that the black sandwich touches the black transfer piece. Fill with transfer buffer.
	#Transfer 4h at 75V (in cold room) or overnight at 35V (room temp).		#Transfer 4h at 75V (in cold room) or overnight at 35V (room temp).
	#Stain for total protein with Revert total protein stain on rocker for 5 minutes --when finished pour total protein stain back in bottle for later use!		#Stain for total protein with Revert total protein stain on rocker for 5 minutes --when finished pour total protein stain back in bottle for later use!
	#Rinse twice in revert wash solution (60ml MeOH, 13.4 ml ~~Aceditc~~ Acid, 126.6 ml Water)		#Rinse twice in revert wash solution (60ml MeOH, 13.4 ml Acetic Acid, 126.6 ml Water)
	#Scan using licor for total protein, which will be used to normalize the blot		#Scan using licor for total protein, which will be used to normalize the blot
	#Rinse nitrocellulose in revert reversal solution for at least 5 and no more than 10 minutes until nitrocellulose appears clear again (.2g NaOH, 60ml MeOH, 140ml Water)		#Rinse nitrocellulose in revert reversal solution for at least 5 and no more than 10 minutes until nitrocellulose appears clear again (.2g NaOH, 60ml MeOH, 140ml Water)