Knight:Electrophoretic mobility shift assay - Revision history

Reshma P. Shetty at 22:34, 5 December 2006

2006-12-05T22:34:05Z

←Older revision		Revision as of 22:34, 5 December 2006
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	==Contact==		==Contact==
	*[[Reshma Shetty]]		*[[Reshma Shetty]]
		+
		+	[[Category:Protocol]]
		+	[[Category:In vitro]]
		+	[[Category:Protein]]
		+	[[Category:DNA]]

Reshma P. Shetty at 20:58, 27 November 2006

2006-11-27T20:58:13Z

←Older revision		Revision as of 20:58, 27 November 2006
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-	<font color=red>~~In progress! Has not been tested~~.</font>	+	<font color=red>This procedure works but likely requires further optimization for best results.</font>

	==Overview==		==Overview==

Reshma P. Shetty: /* Staining the gel */

2006-10-17T22:08:08Z

Staining the gel

←Older revision		Revision as of 22:08, 17 October 2006
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	#*Exact amount depends on size of gel staining tray.		#*Exact amount depends on size of gel staining tray.
	#Incubate ~3 hours on orbital shaker set at 50 rpm, protected from light.		#Incubate ~3 hours on orbital shaker set at 50 rpm, protected from light.
-	#*Don't use a glass container (glass adsorbs the dye).	+	#*Don't use a glass container (glass adsorbs the dye). The lid of a 1000μL pipette tip box seems to be about the right size and work well.
	#*You can leave the gel in stain overnight.		#*You can leave the gel in stain overnight.
	#*Do not dilute the stain.		#*Do not dilute the stain.

Reshma P. Shetty: /* Electrophoresis */

2006-10-17T22:06:48Z

Electrophoresis

←Older revision		Revision as of 22:06, 17 October 2006
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	#*If there is a leak, discard buffer, reseal chamber and try again.		#*If there is a leak, discard buffer, reseal chamber and try again.
	#Fill upper buffer chamber. Buffer level should exceed level of the wells. Requires about 200mL		#Fill upper buffer chamber. Buffer level should exceed level of the wells. Requires about 200mL
-	#Load 6μL 2-log DNA ladder.	+	#Load 3μL 2-log DNA ladder.
	#Load samples.		#Load samples.
-	~~#Load 5μL Mark12 unstained protein molecular weight standard.~~
-	~~#*''Dropped this to 1μL to see if it looks any better.''~~
	#Fill lower buffer chamber at the gap near locking mechanism with 600mL 0.5X TBE running buffer.		#Fill lower buffer chamber at the gap near locking mechanism with 600mL 0.5X TBE running buffer.
	#*''Should the buffer be chilled?''		#*''Should the buffer be chilled?''

Reshma P. Shetty: /* Staining */

2006-10-17T22:06:15Z

Staining

←Older revision		Revision as of 22:06, 17 October 2006
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	**Comes in EMSA kit from Invitrogen ([http://probes.invitrogen.com/media/pis/mp33075.pdf manual], catalog number - [https://catalog.invitrogen.com/index.cfm?fuseaction=viewCatalog.viewProductDetails&productDescription=2538&CMP=LEC-GCMSSEARCH&HQS=EMSA E-33075])		**Comes in EMSA kit from Invitrogen ([http://probes.invitrogen.com/media/pis/mp33075.pdf manual], catalog number - [https://catalog.invitrogen.com/index.cfm?fuseaction=viewCatalog.viewProductDetails&productDescription=2538&CMP=LEC-GCMSSEARCH&HQS=EMSA E-33075])
	**Supposedly different from "normal" SYBR Green nucleic acid stain but not sure this is true.		**Supposedly different from "normal" SYBR Green nucleic acid stain but not sure this is true.
		+	**"Normal" SYBR green nucleic acid stain seems to work just as well.
	*SYPRO Ruby EMSA protein gel stain		*SYPRO Ruby EMSA protein gel stain
	**Light sensitive.		**Light sensitive.

Reshma P. Shetty at 20:39, 5 October 2006

2006-10-05T20:39:32Z

←Older revision		Revision as of 20:39, 5 October 2006
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	#''Before opening'', warm the SYBR green EMSA gel stain concentrate to room temperature.		#''Before opening'', warm the SYBR green EMSA gel stain concentrate to room temperature.
	#Vortex and centrifuge tube.		#Vortex and centrifuge tube.
-	#Dilute 10μL of 10,000X SYBR green EMSA gel stain concentrate into ~~100~~ mL 0.5X TBE buffer and pour into gel staining tray.	+	#Dilute 5μL of 10,000X SYBR green EMSA gel stain concentrate into 50 mL 0.5X TBE buffer and pour into gel staining tray.
	#*Exact amount depends on size of gel staining tray.		#*Exact amount depends on size of gel staining tray.
	#Disconnect electrodes.		#Disconnect electrodes.
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	#Cut to separate gel from bottom lip.		#Cut to separate gel from bottom lip.
	#Flip over and transfer gel to clean staining tray.		#Flip over and transfer gel to clean staining tray.
		+	#*''Use the lid of a 1000μL pipette tip box.''
	#Incubate ~20 mins on orbital shaker set at 50 rpm, protected from light.		#Incubate ~20 mins on orbital shaker set at 50 rpm, protected from light.
	#*Don't use a glass container (glass adsorbs the dye).		#*Don't use a glass container (glass adsorbs the dye).
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	*Promega has a useful [http://www.promega.com/faq/gelshfaq.html FAQ].		*Promega has a useful [http://www.promega.com/faq/gelshfaq.html FAQ].
	*Don't bother running a protein standard. It doesn't come out very well (either faint or blotchy depending on amount).		*Don't bother running a protein standard. It doesn't come out very well (either faint or blotchy depending on amount).
		+	*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.

	==Safety==		==Safety==

Reshma P. Shetty: /* Electrophoresis */

2006-10-05T15:12:22Z

Electrophoresis

←Older revision		Revision as of 15:12, 5 October 2006
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	**1 mM EDTA (free acid)		**1 mM EDTA (free acid)
	**pH ? (do not use acid or base to adjust the pH of the solution)		**pH ? (do not use acid or base to adjust the pH of the solution)
		+	**Can make yourself or buy from Invitrogen (catalog number LC6675)
	*DNA ladder		*DNA ladder
	**Use standard 12μL of 2-log ladder with orange G dye? ''Seems to bleed a bit into acrylamide. Drop the loading volume down to 2 μL. Then the ladder will give an intensity more comparable to the 50ng probe but it still bleeds a bit.''		**Use standard 12μL of 2-log ladder with orange G dye? ''Seems to bleed a bit into acrylamide. Drop the loading volume down to 2 μL. Then the ladder will give an intensity more comparable to the 50ng probe but it still bleeds a bit.''

Reshma P. Shetty: /* Safety */

2006-10-05T15:04:16Z

Safety

←Older revision		Revision as of 15:04, 5 October 2006
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	*Use nitrile gloves while handling acrylamide gels.		*Use nitrile gloves while handling acrylamide gels.
	*TCA is highly corrosive and hazardous. Use proper personal equipment protection.		*TCA is highly corrosive and hazardous. Use proper personal equipment protection.
-	*SYBR green and SYPRO red stains must be disposed of as hazardous waste in separate waste streams. The SYPRO red is mixed with trichloroacetic acid and thus should be marked as ignitable and corrosive and the SYBR green is mixed with DMSO and should be marked toxic. (Kathy Gilbert, EHS)	+	*''SYBR green and SYPRO red stains must be disposed of as hazardous waste in separate waste streams. The SYPRO red is mixed with trichloroacetic acid and thus should be marked as ignitable and corrosive and the SYBR green is mixed with DMSO and should be marked toxic.'' (Kathy Gilbert, EHS)

	==Contact==		==Contact==
	*[[Reshma Shetty]]		*[[Reshma Shetty]]

Reshma P. Shetty: /* Notes */

2006-10-05T15:03:55Z

Notes

←Older revision		Revision as of 15:03, 5 October 2006
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	*''This kit is not sensitive enough for a complex mixture of protein and RNA. This kit has most optimum results when used with a more purified sample. The analysis of a complex solution with low concentrations of the actual target molecule requires more sensitivity than fluorescence can provide.'' From Molecular Probes technical assistance.		*''This kit is not sensitive enough for a complex mixture of protein and RNA. This kit has most optimum results when used with a more purified sample. The analysis of a complex solution with low concentrations of the actual target molecule requires more sensitivity than fluorescence can provide.'' From Molecular Probes technical assistance.
	*Promega has a useful [http://www.promega.com/faq/gelshfaq.html FAQ].		*Promega has a useful [http://www.promega.com/faq/gelshfaq.html FAQ].
		+	*Don't bother running a protein standard. It doesn't come out very well (either faint or blotchy depending on amount).

	==Safety==		==Safety==

Reshma P. Shetty: /* Materials */

2006-10-05T15:02:24Z

Materials

←Older revision		Revision as of 15:02, 5 October 2006
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	===Electrophoresis===		===Electrophoresis===
	*Loading solution		*Loading solution
-	**~~Comes in EMSA kit if we go that route (light~~ sensitive~~, stored~~ at -20°C)	+	**Light sensitive
		+	**Stored at -20°C
		+	**Comes in EMSA kit from Invitrogen ([http://probes.invitrogen.com/media/pis/mp33075.pdf manual], catalog number - [https://catalog.invitrogen.com/index.cfm?fuseaction=viewCatalog.viewProductDetails&productDescription=2538&CMP=LEC-GCMSSEARCH&HQS=EMSA E-33075])
	**Alternatively, Tom thinks we could get away with using our [[Knight:Loading dye\|typical gel loading buffer]].		**Alternatively, Tom thinks we could get away with using our [[Knight:Loading dye\|typical gel loading buffer]].
	**Is this compatible with the DNA retardation gels gels?		**Is this compatible with the DNA retardation gels gels?
-	*[http://www.invitrogen.com/content/sfs/manuals/dnaretardationgel_card.pdf Novex DNA Retardation Gels] ([http://www.invitrogen.com/content/sfs/manuals/electrophoresisguide_man.pdf manual])	+	*[http://www.invitrogen.com/content/sfs/manuals/dnaretardationgel_card.pdf Novex DNA Retardation Gels] ([http://www.invitrogen.com/content/sfs/manuals/electrophoresisguide_man.pdf manual], catalog number - [https://catalog.invitrogen.com/index.cfm?fuseaction=viewCatalog.viewProductDetails&productDescription=32194&CMP=LEC-GCMSSEARCH&HQS=DNA%20retardation EC6365BOX] (10 well) or [https://catalog.invitrogen.com/index.cfm?fuseaction=viewCatalog.viewProductDetails&productDescription=32194&CMP=LEC-GCMSSEARCH&HQS=DNA%20retardation EC63652BOX] (12 well))
-	**Catalog Number - [https://catalog.invitrogen.com/index.cfm?fuseaction=viewCatalog.viewProductDetails&productDescription=32194&CMP=LEC-GCMSSEARCH&HQS=DNA%20retardation EC6365BOX] (10 well) or [https://catalog.invitrogen.com/index.cfm?fuseaction=viewCatalog.viewProductDetails&productDescription=32194&CMP=LEC-GCMSSEARCH&HQS=DNA%20retardation EC63652BOX] (12 well)	+
	*0.5X TBE running buffer		*0.5X TBE running buffer
	**44.5 mM Tris base		**44.5 mM Tris base
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	**pH ? (do not use acid or base to adjust the pH of the solution)		**pH ? (do not use acid or base to adjust the pH of the solution)
	*DNA ladder		*DNA ladder
-	**Use standard 12μL of 2-log ladder with orange G dye? (Seems to bleed a bit into acrylamide). ''Drop the loading volume down to 6 μL. Then the ladder will give an intensity more comparable to the probe~~.''~~	+	**Use standard 12μL of 2-log ladder with orange G dye? ''Seems to bleed a bit into acrylamide. Drop the loading volume down to 2 μL. Then the ladder will give an intensity more comparable to the 50ng probe but it still bleeds a bit.''
-	*Protein molecular weight standard	+
-	*Don't think that people typically run protein molecular weight standards on these gels. (Probably cause you typically only visualize DNA.)	+
-	**Need an unstained protein molecular weight standard (prestained standards interfere with fluorescence of SYPRO Red). Trying Mark12 Unstained Standard. But it ~~might be in the wrong loading buffer. ''Came out somewhat blotchy. Trying just BSA to check protein staining~~.''	+

	===Staining===		===Staining===
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	**Light sensitive		**Light sensitive
	**Stored at -20°C		**Stored at -20°C
		+	**Comes in EMSA kit from Invitrogen ([http://probes.invitrogen.com/media/pis/mp33075.pdf manual], catalog number - [https://catalog.invitrogen.com/index.cfm?fuseaction=viewCatalog.viewProductDetails&productDescription=2538&CMP=LEC-GCMSSEARCH&HQS=EMSA E-33075])
	**Supposedly different from "normal" SYBR Green nucleic acid stain but not sure this is true.		**Supposedly different from "normal" SYBR Green nucleic acid stain but not sure this is true.
	*SYPRO Ruby EMSA protein gel stain		*SYPRO Ruby EMSA protein gel stain
	**Light sensitive.		**Light sensitive.
	**Store at room temperature.		**Store at room temperature.
		+	**Comes in EMSA kit from Invitrogen ([http://probes.invitrogen.com/media/pis/mp33075.pdf manual], catalog number - [https://catalog.invitrogen.com/index.cfm?fuseaction=viewCatalog.viewProductDetails&productDescription=2538&CMP=LEC-GCMSSEARCH&HQS=EMSA E-33075])
	**Supposedly different from "normal" SYPRO Ruby protein stain but not sure this is true.		**Supposedly different from "normal" SYPRO Ruby protein stain but not sure this is true.
	**When mixed with TCA, it is stable for 6 months.		**When mixed with TCA, it is stable for 6 months.