Knight:NuPAGE electrophoresis - Revision history

Jakob Suckale: /* Materials */

Jakob Suckale — Mon, 02 Mar 2009 15:34:58 GMT

Materials

←Older revision		Revision as of 15:34, 2 March 2009
Line 7:		Line 7:
	*NuPAGE Novex 10% Bis-Tris Gel 1.0 mm, 10 well (for small proteins e.g. 13kD) or NuPAGE Novex 4-12% Bis-Tris Gel 1.0 mm, 10 well (for medium proteins e.g. 50-60kD)		*NuPAGE Novex 10% Bis-Tris Gel 1.0 mm, 10 well (for small proteins e.g. 13kD) or NuPAGE Novex 4-12% Bis-Tris Gel 1.0 mm, 10 well (for medium proteins e.g. 50-60kD)
	*[https://catalog.invitrogen.com/index.cfm?fuseaction=viewCatalog.viewProductDetails&productDescription=792& XCell ''SureLock'' Mini-Cell electrophoresis apparatus]		*[https://catalog.invitrogen.com/index.cfm?fuseaction=viewCatalog.viewProductDetails&productDescription=792& XCell ''SureLock'' Mini-Cell electrophoresis apparatus]
-	*20X MES or MOPS Running Buffer	+	*20X MES or [[MOPS]] Running Buffer
	*NuPAGE LDS Sample Buffer (4X)		*NuPAGE LDS Sample Buffer (4X)
	*NuPAGE Reducing Agent (10X)		*NuPAGE Reducing Agent (10X)

Tk: /* Marker Sizes */

Tk — Sat, 13 Sep 2008 00:09:12 GMT

Marker Sizes

←Older revision		Revision as of 00:09, 13 September 2008
Line 93:		Line 93:
	** Aprotinin 9 KD		** Aprotinin 9 KD
	** Insulin B chain 3 KD		** Insulin B chain 3 KD
		+
		+	* [http://www.invitrogen.com/etc/medialib/en/images/mainbody/punchout.Par.89536.Image.-1.-1.1.gif Marker chart for Invitrogen Bis-Tris and Tris-Acetate Nupage gels]

	==Notes==		==Notes==

Tk: /* Marker Sizes */

Tk — Fri, 14 Mar 2008 17:37:40 GMT

Marker Sizes

←Older revision		Revision as of 17:37, 14 March 2008
Line 82:		Line 82:

	===Marker Sizes===		===Marker Sizes===
-	* The SeeBlue Plus 2 marker (blue unless another color is indicated)	+	* The SeeBlue Plus 2 marker (blue unless another color is indicated); 10 μL samples have around 1-2 μg of protein per band
-	** Myosin 188 KD	+	** Myosin 188 KD (MES buffer) 191 KD (MOPS buffer)
-	** Phosphorylase B 98 KD (orange)	+	** Phosphorylase B 98 KD (MES buffer) 97 KD (MOPS buffer) (orange)
-	** BSA 62 KD	+	** BSA 62 KD (MES buffer) 64 KD (MOPS buffer)
-	** Glutamic dehydrogenase 49 KD	+	** Glutamic dehydrogenase 49 KD (MES buffer) 51 KD (MOPS buffer)
-	** Alcohol dehydrogenase 38 KD	+	** Alcohol dehydrogenase 38 KD (MES buffer) 39 KD (MOPS buffer)
	** Carbonic anhydrase 28 KD		** Carbonic anhydrase 28 KD
-	** Myoglobin 17 KD (purple)	+	** Myoglobin 17 KD (MES buffer) 19 KD (MOPS buffer) (purple)
	** Lysozyme 14 KD		** Lysozyme 14 KD
	** Aprotinin 9 KD		** Aprotinin 9 KD
	** Insulin B chain 3 KD		** Insulin B chain 3 KD
-

	==Notes==		==Notes==

Tk: /* Notes */

Tk — Mon, 10 Mar 2008 03:08:02 GMT

Notes

←Older revision		Revision as of 03:08, 10 March 2008
Line 98:		Line 98:
	*To date, the best staining tray I've found is the lid of a 1000μL pipette tip box. Then use a piece of mesh that just fits inside the lide to either keep the gel in place while changing solutions or to move the gel to and from the light box. This method requires smaller volumes of stain than the staining tray from Invitrogen.		*To date, the best staining tray I've found is the lid of a 1000μL pipette tip box. Then use a piece of mesh that just fits inside the lide to either keep the gel in place while changing solutions or to move the gel to and from the light box. This method requires smaller volumes of stain than the staining tray from Invitrogen.
	*There are several ways to screw up this protocol, I list them here in the hopes that it may help people avoid these trivial mistakes.		*There are several ways to screw up this protocol, I list them here in the hopes that it may help people avoid these trivial mistakes.
		+	*#Failing to remove the seal at the bottom of the precast gel
	*#The inner and outer chambers of running buffer are not sealed from one another.		*#The inner and outer chambers of running buffer are not sealed from one another.
	*#Reverse the electrodes in the power supply.		*#Reverse the electrodes in the power supply.

Tk at 03:06, 10 March 2008

Tk — Mon, 10 Mar 2008 03:06:51 GMT

←Older revision		Revision as of 03:06, 10 March 2008
Line 79:		Line 79:

	See [[Knight:NuPAGE electrophoresis/Gel drying]] for instructions on how to dry and preserve the gel.		See [[Knight:NuPAGE electrophoresis/Gel drying]] for instructions on how to dry and preserve the gel.
		+
		+
		+	===Marker Sizes===
		+	* The SeeBlue Plus 2 marker (blue unless another color is indicated)
		+	** Myosin 188 KD
		+	** Phosphorylase B 98 KD (orange)
		+	** BSA 62 KD
		+	** Glutamic dehydrogenase 49 KD
		+	** Alcohol dehydrogenase 38 KD
		+	** Carbonic anhydrase 28 KD
		+	** Myoglobin 17 KD (purple)
		+	** Lysozyme 14 KD
		+	** Aprotinin 9 KD
		+	** Insulin B chain 3 KD
		+

	==Notes==		==Notes==

Reshma P. Shetty at 21:37, 23 February 2008

Reshma P. Shetty — Sat, 23 Feb 2008 21:37:52 GMT

←Older revision		Revision as of 21:37, 23 February 2008
Line 11:		Line 11:
	*NuPAGE Reducing Agent (10X)		*NuPAGE Reducing Agent (10X)
	*NuPAGE Antioxidant		*NuPAGE Antioxidant
-	*SeeBlue Plus2 Pre-Stained Standard	+	*[https://catalog.invitrogen.com/productImages/1900/1896.GIF SeeBlue Plus2 Pre-Stained Standard]
	*[http://www.invitrogen.com/content.cfm?pageid=9869 Invitrogen SimplyBlue SafeStain]		*[http://www.invitrogen.com/content.cfm?pageid=9869 Invitrogen SimplyBlue SafeStain]

Reshma P. Shetty: /* References */

Reshma P. Shetty — Thu, 23 Aug 2007 16:24:18 GMT

References

←Older revision		Revision as of 16:24, 23 August 2007
Line 98:		Line 98:
	</biblio>		</biblio>

-	[[Category:Protein]] [[Category:In vitro]]	+	[[Category:Protein]] [[Category:In vitro]] [[Category:Protocol]]

Reshma P. Shetty: /* Notes */

Reshma P. Shetty — Wed, 25 Jul 2007 19:52:10 GMT

Notes

←Older revision		Revision as of 19:52, 25 July 2007
Line 82:		Line 82:
	==Notes==		==Notes==
	*To date, the best staining tray I've found is the lid of a 1000μL pipette tip box. Then use a piece of mesh that just fits inside the lide to either keep the gel in place while changing solutions or to move the gel to and from the light box. This method requires smaller volumes of stain than the staining tray from Invitrogen.		*To date, the best staining tray I've found is the lid of a 1000μL pipette tip box. Then use a piece of mesh that just fits inside the lide to either keep the gel in place while changing solutions or to move the gel to and from the light box. This method requires smaller volumes of stain than the staining tray from Invitrogen.
		+	*There are several ways to screw up this protocol, I list them here in the hopes that it may help people avoid these trivial mistakes.
		+	*#The inner and outer chambers of running buffer are not sealed from one another.
		+	*#Reverse the electrodes in the power supply.
		+	*#Putting the gel in backwards in the apparatus.

	==Safety==		==Safety==

Reshma P. Shetty: /* Materials */

Reshma P. Shetty — Fri, 08 Jun 2007 16:37:00 GMT

Materials

←Older revision		Revision as of 16:37, 8 June 2007
Line 5:		Line 5:

	==Materials==		==Materials==
-	*NuPAGE Novex 10% Bis-Tris Gel 1.0 mm, 10 well	+	*NuPAGE Novex 10% Bis-Tris Gel 1.0 mm, 10 well (for small proteins e.g. 13kD) or NuPAGE Novex 4-12% Bis-Tris Gel 1.0 mm, 10 well (for medium proteins e.g. 50-60kD)
	*[https://catalog.invitrogen.com/index.cfm?fuseaction=viewCatalog.viewProductDetails&productDescription=792& XCell ''SureLock'' Mini-Cell electrophoresis apparatus]		*[https://catalog.invitrogen.com/index.cfm?fuseaction=viewCatalog.viewProductDetails&productDescription=792& XCell ''SureLock'' Mini-Cell electrophoresis apparatus]
	*20X MES or MOPS Running Buffer		*20X MES or MOPS Running Buffer

Reshma P. Shetty: /* Purpose */

Reshma P. Shetty — Fri, 08 Jun 2007 16:35:45 GMT

Purpose

←Older revision		Revision as of 16:35, 8 June 2007
Line 3:		Line 3:
	==Purpose==		==Purpose==
	To run a denaturing protein gel. Note that a preferable version of this protocol is available at [[Sauer:bis-Tris SDS-PAGE, the very best]] (recommended by Kathleen). But since we seem to already have the Invitrogen kit contents in lab, this protocol describes use of the Invitrogen system.		To run a denaturing protein gel. Note that a preferable version of this protocol is available at [[Sauer:bis-Tris SDS-PAGE, the very best]] (recommended by Kathleen). But since we seem to already have the Invitrogen kit contents in lab, this protocol describes use of the Invitrogen system.
-
-	~~Note that the gel and running buffer were chosen for a 13kD protein.~~

	==Materials==		==Materials==