Acrylamide gels - Revision history

Reshma P. Shetty: Acrylamide Gels moved to Acrylamide gels: capitalization convention

2006-08-10T21:10:38Z

Acrylamide Gels moved to Acrylamide gels: capitalization convention

←Older revision

Revision as of 21:10, 10 August 2006

Jenny T Nguyen at 19:27, 10 July 2006

2006-07-10T19:27:07Z

←Older revision		Revision as of 19:27, 10 July 2006
Line 4:		Line 4:
	[[Sauer:bis-Tris SDS-PAGE, the very best]]<br>		[[Sauer:bis-Tris SDS-PAGE, the very best]]<br>
	[[Keating:Experimental Protocols:SDS-PAGE]]<br>		[[Keating:Experimental Protocols:SDS-PAGE]]<br>
		+
		+
		+	[[Category:Protocol]]
		+	[[Category:Protein]]
		+	[[Category:In vitro]]

Barry Canton at 17:14, 17 January 2006

2006-01-17T17:14:09Z

←Older revision		Revision as of 17:14, 17 January 2006
Line 1:		Line 1:
-	~~Here are some links to some SDS page protocols.~~	+	==Lab Protocols==

	[[Endy:Tris-Tricine Acrylamide Gels]]<br>		[[Endy:Tris-Tricine Acrylamide Gels]]<br>
	[[Sauer:bis-Tris SDS-PAGE, the very best]]<br>		[[Sauer:bis-Tris SDS-PAGE, the very best]]<br>
	[[Keating:Experimental Protocols:SDS-PAGE]]<br>		[[Keating:Experimental Protocols:SDS-PAGE]]<br>

Skosuri at 16:12, 19 September 2005

2005-09-19T16:12:41Z

←Older revision		Revision as of 16:12, 19 September 2005
Line 1:		Line 1:
-	[[Endy:Tris-Tricine Acrylamide Gels]]	+	Here are some links to some SDS page protocols.
		+
		+	[[Endy:Tris-Tricine Acrylamide Gels]]<br>
		+	[[Sauer:bis-Tris SDS-PAGE, the very best]]<br>
		+	[[Keating:Experimental Protocols:SDS-PAGE]]<br>

Skosuri at 16:05, 19 September 2005

2005-09-19T16:05:53Z

←Older revision		Revision as of 16:05, 19 September 2005
Line 1:		Line 1:
-	~~==Pouring~~ Tris-Tricine Acrylamide Gels==	+	[[Endy:Tris-Tricine Acrylamide Gels]]
-	~~Protocol by [[Jennifer Braff\|jcb~~]]	+
-	~~==Running Gel (10%)==~~	+
-	*4.9 mL 30% acrylamide/0.8% bisacrylamide (37.5:1)	+
-	*5 mL Tris-Cl/SDS pH 8.45	+
-	*1.15 mL MilliQ water	+
-	*3.95 mL 40% glycerol	+
-		+
-	*25 uL 10% APS (made fresh)	+
-	*5 uL TEMED	+
-	~~==Stacking Gel (4%)==~~	+
-	*0.81 mL 30% acrylamide/0.8% bisacrylamide	+
-	*1.55 mL Tris-Cl/SDS pH 8.45	+
-	*3.89 mL MilliQ water	+
-	*40 uL saturated bromophenol blue (optional)	+
-		+
-	*37.5 uL 10% APS (0.1g ammonium persulfate/mL H20)	+
-	*17.5 uL TEMED	+
-		+
-		+
-	~~Add APS and TEMED immediately before pouring.~~	+
-		+
-	Pour running (separating) gel (use 20G 1.5 syringe) to 1 cm below bottom of comb (mark short plate) and overlay with isopropanol. Set for 1 hr, rinse with water (buffer might be better), and pour stacking gel. Insert combs, being careful to avoid bubbles. Let set, wrap in damp paper towels, and store 4 C for up to one week.	+
-		+
-	15 well, .75 mm gels hold about 15 uL sample per lane. To run gels (MiniProtean III), put 200 mL Anode Buffer in lower/outer chamber and 125 mL Cathode Buffer in inner/upper chamber. Remove combs after adding running buffers. Use a syringe or x-long gel-loading pipette tip to flush out each well gently with Cathode Buffer to remove any unpolymerized acrylamide.	+
-		+
-	~~Run at about 80 V for a couple of hours.~~	+
-		+
-	~~== Tris-Tricine Cathode Buffer==~~	+
-	*12.22 g Tris (0.1 M)	+
-	*17.92 g Tricine (0.1 M)	+
-	*1 g SDS (0.1%)	+
-	*to 1 L with MilliQ H2O (no need to pH)	+
-	~~== Tris-Tricine Anode Buffer (5L)==~~	+
-	*121 g Tris (0.2 M)	+
-	*500 mL MilliQ H2O	+
-	*pH to 8.9 with 6N HCl (about 30 mL)	+
-	*to 5 L with MilliQ	+
-	~~==Tris-Cl/SDS pH 8.45==~~	+
-	*182 g Tris base in 300 mL MilliQ H2O (Tris is 121.14 g/mol) (hard to dissolve)	+
-	*to pH 8.45 with 3N Hcl	+
-	*MilliQ H2O to 500 mL	+
-	*Filter 0.45 um	+
-	*Add 1.5 g SDS	+

Skosuri at 15:52, 19 September 2005

2005-09-19T15:52:24Z

←Older revision		Revision as of 15:52, 19 September 2005
Line 1:		Line 1:
	==Pouring Tris-Tricine Acrylamide Gels==		==Pouring Tris-Tricine Acrylamide Gels==
-	jcb	+	Protocol by [[Jennifer Braff\|jcb]]
	==Running Gel (10%)==		==Running Gel (10%)==
	*4.9 mL 30% acrylamide/0.8% bisacrylamide (37.5:1)		*4.9 mL 30% acrylamide/0.8% bisacrylamide (37.5:1)

Jcbraff at 22:33, 16 July 2005

2005-07-16T22:33:09Z

New page

==Pouring Tris-Tricine Acrylamide Gels==
jcb
==Running Gel (10%)==
*4.9 mL 30% acrylamide/0.8% bisacrylamide (37.5:1)
*5 mL Tris-Cl/SDS pH 8.45
*1.15 mL MilliQ water
*3.95 mL 40% glycerol

*25 uL 10% APS (made fresh)
*5 uL TEMED
==Stacking Gel (4%)==
*0.81 mL 30% acrylamide/0.8% bisacrylamide
*1.55 mL Tris-Cl/SDS pH 8.45
*3.89 mL MilliQ water
*40 uL saturated bromophenol blue (optional)

*37.5 uL 10% APS (0.1g ammonium persulfate/mL H20)
*17.5 uL TEMED

Add APS and TEMED immediately before pouring.

Pour running (separating) gel (use 20G 1.5 syringe) to 1 cm below bottom of comb (mark short plate) and overlay with isopropanol. Set for 1 hr, rinse with water (buffer might be better), and pour stacking gel. Insert combs, being careful to avoid bubbles. Let set, wrap in damp paper towels, and store 4 C for up to one week.

15 well, .75 mm gels hold about 15 uL sample per lane. To run gels (MiniProtean III), put 200 mL Anode Buffer in lower/outer chamber and 125 mL Cathode Buffer in inner/upper chamber. Remove combs after adding running buffers. Use a syringe or x-long gel-loading pipette tip to flush out each well gently with Cathode Buffer to remove any unpolymerized acrylamide.

Run at about 80 V for a couple of hours.

== Tris-Tricine Cathode Buffer==
*12.22 g Tris (0.1 M)
*17.92 g Tricine (0.1 M)
*1 g SDS (0.1%)
*to 1 L with MilliQ H2O (no need to pH)
== Tris-Tricine Anode Buffer (5L)==
*121 g Tris (0.2 M)
*500 mL MilliQ H2O
*pH to 8.9 with 6N HCl (about 30 mL)
*to 5 L with MilliQ
==Tris-Cl/SDS pH 8.45==
*182 g Tris base in 300 mL MilliQ H2O (Tris is 121.14 g/mol) (hard to dissolve)
*to pH 8.45 with 3N Hcl
*MilliQ H2O to 500 mL
*Filter 0.45 um
*Add 1.5 g SDS