Shreffler: SDS-PAGE - Revision history

Alex Ma: /* Running Gel */

Alex Ma — Thu, 22 Mar 2012 20:00:12 GMT

Running Gel

←Older revision		Revision as of 20:00, 22 March 2012
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	#Set aside 200 ml of Running Buffer and add 0.5 ml Antioxidant.		#Set aside 200 ml of Running Buffer and add 0.5 ml Antioxidant.
	#Remove precast 4-12% Bis-Tris gels from packages, carefully and over a sink (pouch contains liquid). Pull off white stripe at bottom and carefully remove plastic comb from gel. Gently wash cassette and (esp. wells) under DI water (can apply gentle centrifugal force to eject residual liquid from wells so that they can be washed).		#Remove precast 4-12% Bis-Tris gels from packages, carefully and over a sink (pouch contains liquid). Pull off white stripe at bottom and carefully remove plastic comb from gel. Gently wash cassette and (esp. wells) under DI water (can apply gentle centrifugal force to eject residual liquid from wells so that they can be washed).
		+	#Place gel cassette on tube rack (stabilize vertically) and fill wells with standard (5 µl) and samples of interest - can run varying volumes of loading solution to vary protein quantity (load 2 µl for 1 µg protein, 10 µl for 5 µg protein).
	#Lower buffer core into cell and insert gel cassettes on both sides, such that the shorter well side faces inward (if only running one gel, use a dummy cassette). Insert tension wedge and lock in place.		#Lower buffer core into cell and insert gel cassettes on both sides, such that the shorter well side faces inward (if only running one gel, use a dummy cassette). Insert tension wedge and lock in place.
	#Fill buffer core with the 200 ml of Running Buffer + Antioxidant. Pour slowly and make sure that core does NOT leak into outer cell.		#Fill buffer core with the 200 ml of Running Buffer + Antioxidant. Pour slowly and make sure that core does NOT leak into outer cell.
	#Fill outer space (between cell and core) with Running Buffer (no Antioxidant) until level is sufficient (~600 ml)		#Fill outer space (between cell and core) with Running Buffer (no Antioxidant) until level is sufficient (~600 ml)
-	~~#Fill wells with standard (5 µl) and samples of interest - can run varying volumes of loading solution to vary protein quantity (load 2 µl for 1 µg protein, 10 µl for 5 µg protein).~~
	#Match electrode ends and turn voltage source on. Set to 100V to begin with, but can increase to 150V when gel is halfway done running (gel has density gradient, so bands will run slower the further down they are).		#Match electrode ends and turn voltage source on. Set to 100V to begin with, but can increase to 150V when gel is halfway done running (gel has density gradient, so bands will run slower the further down they are).
	#Stop running the gel when blue indicator bands are almost at bottom.		#Stop running the gel when blue indicator bands are almost at bottom.

Alex Ma: /* Procedure */

Alex Ma — Thu, 22 Mar 2012 19:49:56 GMT

Procedure

←Older revision		Revision as of 19:49, 22 March 2012
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	===Running Gel===		===Running Gel===

-	#Make 1X	+	#Make MES SDS Running Buffer 1X using 20X stock (~one liter should be more than enough).
		+	#Set aside 200 ml of Running Buffer and add 0.5 ml Antioxidant.
	#Remove precast 4-12% Bis-Tris gels from packages, carefully and over a sink (pouch contains liquid). Pull off white stripe at bottom and carefully remove plastic comb from gel. Gently wash cassette and (esp. wells) under DI water (can apply gentle centrifugal force to eject residual liquid from wells so that they can be washed).		#Remove precast 4-12% Bis-Tris gels from packages, carefully and over a sink (pouch contains liquid). Pull off white stripe at bottom and carefully remove plastic comb from gel. Gently wash cassette and (esp. wells) under DI water (can apply gentle centrifugal force to eject residual liquid from wells so that they can be washed).
	#Lower buffer core into cell and insert gel cassettes on both sides, such that the shorter well side faces inward (if only running one gel, use a dummy cassette). Insert tension wedge and lock in place.		#Lower buffer core into cell and insert gel cassettes on both sides, such that the shorter well side faces inward (if only running one gel, use a dummy cassette). Insert tension wedge and lock in place.
-	#	+	#Fill buffer core with the 200 ml of Running Buffer + Antioxidant. Pour slowly and make sure that core does NOT leak into outer cell.
-		+	#Fill outer space (between cell and core) with Running Buffer (no Antioxidant) until level is sufficient (~600 ml)
-	Fill ~~the~~ buffer core with 200 ~~mL 1X RB~~. ~~Make~~ sure ~~this section~~ does NOT leak ~~before adding liquid to the outside. Carefully remove the comb and pipette out any air bubbles. Then, fill~~ outer ~~part of case with 600 mL of RB~~.	+	#Fill wells with standard (5 µl) and samples of interest - can run varying volumes of loading solution to vary protein quantity (load 2 µl for 1 µg protein, 10 µl for 5 µg protein).
-	Fill ~~the wells~~ with ~~samples:~~	+	#Match electrode ends and turn voltage source on. Set to 100V to begin with, but can increase to 150V when gel is halfway done running (gel has density gradient, so bands will run slower the further down they are).
-	~~Add 10 μL MW markers to well~~ #1.	+	#Stop running the gel when blue indicator bands are almost at bottom.
-	Fill ~~remaining 11~~ wells with ~~10 μL~~ of ~~prepared protein sample~~ solution.	+	#Remove gel cassette from gel box, and carefully crack edges open using metal spatula (knife works too). Start at corners, inserting tool and twisting until separation occurs, then work way down sides. Do not insert tool deep enough to touch gel, or else gel will rip when separating cassette sides.
-	~~Fill each well slowly. Try~~ to ~~avoid bubbles in wells that may cause overflow into other wells~~	+	#Fill deep tray with DI water and open gel cassette while submerged under water. Gel should be stuck on one side of the cassette. Carefully trim off bottom blue bands, and the wells on top. Then, carefully remove gel.
-	~~When working with~~ 2 ~~gel plates~~, ~~make sure to label the outside of the container~~.	+	#Place gel in pipette box cover with DI water and let shake at very low speed. Replace DI water every five minutes for fifteen minutes of washing.
-	Match ~~the + and -~~electrode ends ~~(same colors)~~	+	#Pour off DI water completely and stain gel with 15 ml of SimplyBlue SafeStain. Set on shaker (very low speed) for one hour.
-	~~Turn~~ on ~~(be sure Range Select is OFF)~~. Set ~~Range~~ to ~~200V/400mA~~, ~~then hit Start.~~	+	#Pour off blue stain completely and wash gel with DI water, shaking at very low speed for two hours. Replace DI water every 20 minutes or so.
-	~~Run for 35 min. When~~ done, ~~then turn off~~.	+	#Carefully place gel between transparent folder sheet. Place against white paper backdrop and scan or photograph as desired.
-	~~Gel stain:~~	+
-	~~Take~~ gel cassette ~~out of Invitrogen~~ gel box.	+
-	~~Fill microwave safe glass container with MilliQ water~~ enough as to ~~immerse~~ gel.	+
-	~~Crack open~~ gel cassette ~~and place gel in microwave safe glass container~~.	+
-	~~Microwave~~ gel ~~for two minutes or until~~ water ~~boils~~.	+
-	~~Place container~~ on ~~shaker until cool~~. ~~Then decant~~ the ~~water~~. ~~Rinse one more time with MilliQ water~~.	+
-	~~Add Denville Blue Protein Stain to~~ gel ~~container~~ and ~~immerse gel~~.	+
-	~~Microwave gel~~ for ~~two~~ minutes ~~or until protein stain solution boils~~ and ~~place~~ on shaker ~~until cool~~.	+
-	~~Decant gel~~ stain ~~into waste container,~~ and ~~rinse~~ gel with ~~MilliQ~~ water.	+
-	~~Add MilliQ~~ water ~~to immerse gel and microwave for two~~ minutes ~~and then shake until cool~~.	+
-	Carefully place gel ~~into plastic gel box filled with water~~.	+

	==Discussion==		==Discussion==

Alex Ma: /* Materials */

Alex Ma — Thu, 22 Mar 2012 18:53:13 GMT

Materials

←Older revision		Revision as of 18:53, 22 March 2012
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	*NuPAGE Antioxidant (Invitrogen NP0005)		*NuPAGE Antioxidant (Invitrogen NP0005)
	*NuPAGE Reducing Agent (Invitrogen NP0004)		*NuPAGE Reducing Agent (Invitrogen NP0004)
		+	*Novex Sharp Pre-Stained Protein Standard (Invitrogen 57318)

	==Procedure==		==Procedure==

Alex Ma: /* Procedure */

Alex Ma — Thu, 22 Mar 2012 18:45:39 GMT

Procedure

←Older revision		Revision as of 18:45, 22 March 2012
Line 27:		Line 27:

	===Running Gel===		===Running Gel===
		+
		+	#Make 1X
		+	#Remove precast 4-12% Bis-Tris gels from packages, carefully and over a sink (pouch contains liquid). Pull off white stripe at bottom and carefully remove plastic comb from gel. Gently wash cassette and (esp. wells) under DI water (can apply gentle centrifugal force to eject residual liquid from wells so that they can be washed).
		+	#Lower buffer core into cell and insert gel cassettes on both sides, such that the shorter well side faces inward (if only running one gel, use a dummy cassette). Insert tension wedge and lock in place.
		+	#
		+
		+	Fill the buffer core with 200 mL 1X RB. Make sure this section does NOT leak before adding liquid to the outside. Carefully remove the comb and pipette out any air bubbles. Then, fill outer part of case with 600 mL of RB.
		+	Fill the wells with samples:
		+	Add 10 μL MW markers to well #1.
		+	Fill remaining 11 wells with 10 μL of prepared protein sample solution.
		+	Fill each well slowly. Try to avoid bubbles in wells that may cause overflow into other wells
		+	When working with 2 gel plates, make sure to label the outside of the container.
		+	Match the + and -electrode ends (same colors)
		+	Turn on (be sure Range Select is OFF). Set Range to 200V/400mA, then hit Start.
		+	Run for 35 min. When done, then turn off.
		+	Gel stain:
		+	Take gel cassette out of Invitrogen gel box.
		+	Fill microwave safe glass container with MilliQ water enough as to immerse gel.
		+	Crack open gel cassette and place gel in microwave safe glass container.
		+	Microwave gel for two minutes or until water boils.
		+	Place container on shaker until cool. Then decant the water. Rinse one more time with MilliQ water.
		+	Add Denville Blue Protein Stain to gel container and immerse gel.
		+	Microwave gel for two minutes or until protein stain solution boils and place on shaker until cool.
		+	Decant gel stain into waste container, and rinse gel with MilliQ water.
		+	Add MilliQ water to immerse gel and microwave for two minutes and then shake until cool.
		+	Carefully place gel into plastic gel box filled with water.

	==Discussion==		==Discussion==

Alex Ma: /* Procedure */

Alex Ma — Thu, 22 Mar 2012 18:36:03 GMT

Procedure

←Older revision		Revision as of 18:36, 22 March 2012
Line 20:		Line 20:

	#Make a 20 µl loading solution for each protein consisting of:		#Make a 20 µl loading solution for each protein consisting of:
-	**5 µL LDS Sample Buffer (4X)	+	#*5 µL LDS Sample Buffer (4X)
-	**2 µL Reducing Agent	+	#*2 µL Reducing Agent
-	**Volume containing 10 µg protein (e.g. if protein stock solution is 1 µg/µl, add 10 µl)	+	#*Volume containing 10 µg protein (e.g. if protein stock solution is 1 µg/µl, add 10 µl)
-	**Bring up to 20 µl total with PBS.	+	#*Bring up to 20 µl total with PBS.
	#Heat samples on heating block (or water bath) 70°C for 10 mins (protein denaturing and SDS binding).		#Heat samples on heating block (or water bath) 70°C for 10 mins (protein denaturing and SDS binding).

Alex Ma at 18:34, 22 March 2012

Alex Ma — Thu, 22 Mar 2012 18:34:40 GMT

←Older revision		Revision as of 18:34, 22 March 2012
Line 20:		Line 20:

	#Make a 20 µl loading solution for each protein consisting of:		#Make a 20 µl loading solution for each protein consisting of:
-	*5 µL LDS Sample Buffer (4X)	+	**5 µL LDS Sample Buffer (4X)
-	*2 µL Reducing Agent	+	**2 µL Reducing Agent
-	*Volume containing 10 µg protein (e.g. if protein stock solution is 1 µg/µl, add 10 µl)	+	**Volume containing 10 µg protein (e.g. if protein stock solution is 1 µg/µl, add 10 µl)
-	*Bring up to 20 µl total with PBS.	+	**Bring up to 20 µl total with PBS.
	#Heat samples on heating block (or water bath) 70°C for 10 mins (protein denaturing and SDS binding).		#Heat samples on heating block (or water bath) 70°C for 10 mins (protein denaturing and SDS binding).

Alex Ma at 18:33, 22 March 2012

Alex Ma — Thu, 22 Mar 2012 18:33:59 GMT

←Older revision		Revision as of 18:33, 22 March 2012
Line 19:		Line 19:
	===Preparing Samples===		===Preparing Samples===

-	~~1. We want to~~	+	#Make a 20 µl loading solution for each protein consisting of:
-	~~Add~~ 5 ~~μL 4X Reducing~~ Buffer (RB) ~~to 15μL~~ protein ~~solution~~. ~~Vortex sample~~ to ~~mix~~.	+	*5 µL LDS Sample Buffer (4X)
-	Heat samples ~~at 70 °C~~ for 10 mins.	+	*2 µL Reducing Agent
-	~~Spin samples.~~	+	*Volume containing 10 µg protein (e.g. if protein stock solution is 1 µg/µl, add 10 µl)
		+	*Bring up to 20 µl total with PBS.
		+	#Heat samples on heating block (or water bath) 70°C for 10 mins (protein denaturing and SDS binding).
		+
		+	===Running Gel===

	==Discussion==		==Discussion==

Alex Ma: New page: ==Overview== From wikipedia: "SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis, describes a technique widely used in biochemistry, forensics, genetics and molecular bio...

Alex Ma — Thu, 22 Mar 2012 18:15:29 GMT

New page: ==Overview== From wikipedia: "SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis, describes a technique widely used in biochemistry, forensics, genetics and molecular bio...

New page

==Overview==

From wikipedia: "SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis, describes a technique widely used in biochemistry, forensics, genetics and molecular biology to separate proteins according to their electrophoretic mobility (a function of the length of a polypeptide chain and its charge). In most proteins, the binding of SDS to the polypeptide chain imparts an even distribution of charge per unit mass, thereby resulting in a fractionation by approximate size during electrophoresis."

We use the Invitrogen NuPAGE system.

==Materials==

*Appropriate gelbox/voltage source
*NuPAGE 4-12% BIS-TRIS Gel (Invitrogen NP0322BOX)
*NuPAGE MES SDS Running Buffer (20x) (Invitrogen NP0002)
*NuPAGE SimplyBlue SafeStain (Invitrogen LC6060)
*NuPAGE LDS Sample Buffer (4X) (Invitrogen NP0007)
*NuPAGE Antioxidant (Invitrogen NP0005)
*NuPAGE Reducing Agent (Invitrogen NP0004)

==Procedure==

===Preparing Samples===

1. We want to
Add 5 μL 4X Reducing Buffer (RB) to 15μL protein solution. Vortex sample to mix.
Heat samples at 70 °C for 10 mins.
Spin samples.

==Discussion==
[[Talk:{{PAGENAME}}|discuss this protocol]]

==Contact==
*Who has experience with this protocol?
*[[Special:Emailuser/Alex Ma|Email Alex Ma through OpenWetWare]]

[[Category:Protocol]]