Lidstrom: SDS-PAGE - Revision history

Janet B. Matsen: /* Pour the Resolving Gel */

2013-05-14T19:09:30Z

Pour the Resolving Gel

←Older revision		Revision as of 19:09, 14 May 2013
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	* Carefully layer 50%EtOH 50% ddH2O on top of the gel to prevent the top of the gel from drying out		* Carefully layer 50%EtOH 50% ddH2O on top of the gel to prevent the top of the gel from drying out
	** It is very possible to ruin the desired evenness of the resolving gel by squirting this mixture in too fast. Janet has done it!		** It is very possible to ruin the desired evenness of the resolving gel by squirting this mixture in too fast. Janet has done it!
-	* ~~Let dry for~~ 40 minutes.	+	* Wait 40 minutes for anaerobic polymerization.
	* Rinse ethanol/water mix off or you will get bubbles in the gel.		* Rinse ethanol/water mix off or you will get bubbles in the gel.
	** Rinse the gel aggressively by flushing with tap water from the sink.		** Rinse the gel aggressively by flushing with tap water from the sink.
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	** Prop the gel against the casting stand on its side so the liquid drains to one edge. Remove this with paper towel.		** Prop the gel against the casting stand on its side so the liquid drains to one edge. Remove this with paper towel.
	* Store at 4 deg C wrapped in a wet paper towel and saran wrap if you're not going to use it right away.		* Store at 4 deg C wrapped in a wet paper towel and saran wrap if you're not going to use it right away.
-	** You can also store in the fridge after pouring the stacking gel.	+	** You can also store in the fridge after pouring the stacking gel.

	=== '''Pour the Stacking Gel'''===		=== '''Pour the Stacking Gel'''===

Nicole Smalley: /* Prepping Gels */

2013-03-27T00:55:52Z

Prepping Gels

←Older revision		Revision as of 00:55, 27 March 2013
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	** entire sample will leak out		** entire sample will leak out

-	**If using commercially prepared gels, don't forget to remove the tape on the bottom of the gel! If you load your samples and turn on the power and see voltage but no current, check for tape removal!	+	*If using commercially prepared gels, don't forget to remove the tape on the bottom of the gel! If you load your samples and turn on the power and see voltage but no current, check for tape removal (duh, Nicole)!

	=== Preparing Samples ===		=== Preparing Samples ===

Nicole Smalley: /* Prepping Gels */

2013-03-27T00:55:02Z

Prepping Gels

←Older revision		Revision as of 00:55, 27 March 2013
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	*taking the gel off the casting stand before it has polymerized		*taking the gel off the casting stand before it has polymerized
	** entire sample will leak out		** entire sample will leak out
		+
		+	**If using commercially prepared gels, don't forget to remove the tape on the bottom of the gel! If you load your samples and turn on the power and see voltage but no current, check for tape removal!

	=== Preparing Samples ===		=== Preparing Samples ===

Janet B. Matsen: /* Running Gels */

2013-01-04T21:56:45Z

Running Gels

←Older revision		Revision as of 21:56, 4 January 2013
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	* not having the lid to the running unit on all the way. --> poor electrical contact & blurry bands		* not having the lid to the running unit on all the way. --> poor electrical contact & blurry bands
	* Using resolving gel Tris Buffer instead of the intended electrode buffer.		* Using resolving gel Tris Buffer instead of the intended electrode buffer.
-	** The results of said mistake: [[image:SDS-PAGE gel run with resolving gel buffer instead of the proper electrode buffer.jpg\|thumb\|upright=1.5\|center\|SDS-PAGE gel run with resolving gel buffer instead of the proper electrode buffer]]	+	** The results of said mistake: [[User:Janet B. Matsen\|JM 2013/01/04]] [[image:SDS-PAGE gel run with resolving gel buffer instead of the proper electrode buffer.jpg\|thumb\|upright=1.5\|center\|SDS-PAGE gel run with resolving gel buffer instead of the proper electrode buffer]]

	=== Visualizing Gels ===		=== Visualizing Gels ===

Janet B. Matsen: /* Running Gels */

2013-01-04T21:56:04Z

Running Gels

←Older revision		Revision as of 21:56, 4 January 2013
Line 160:		Line 160:
	* not having the lid to the running unit on all the way. --> poor electrical contact & blurry bands		* not having the lid to the running unit on all the way. --> poor electrical contact & blurry bands
	* Using resolving gel Tris Buffer instead of the intended electrode buffer.		* Using resolving gel Tris Buffer instead of the intended electrode buffer.
-	** The results of said mistake: [[image:SDS-PAGE gel run with resolving gel buffer instead of the proper electrode buffer.jpg\|thumb\|upright=3.0\|center\|SDS-PAGE gel run with resolving gel buffer instead of the proper electrode buffer]]	+	** The results of said mistake: [[image:SDS-PAGE gel run with resolving gel buffer instead of the proper electrode buffer.jpg\|thumb\|upright=1.5\|center\|SDS-PAGE gel run with resolving gel buffer instead of the proper electrode buffer]]

	=== Visualizing Gels ===		=== Visualizing Gels ===

Janet B. Matsen: /* Running Gels */

2013-01-04T21:53:59Z

Running Gels

←Older revision		Revision as of 21:53, 4 January 2013
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	* Using Tris-HCl instead of Tris in the electrode buffer		* Using Tris-HCl instead of Tris in the electrode buffer
	* not having the lid to the running unit on all the way. --> poor electrical contact & blurry bands		* not having the lid to the running unit on all the way. --> poor electrical contact & blurry bands
		+	* Using resolving gel Tris Buffer instead of the intended electrode buffer.
		+	** The results of said mistake: [[image:SDS-PAGE gel run with resolving gel buffer instead of the proper electrode buffer.jpg\|thumb\|upright=3.0\|center\|SDS-PAGE gel run with resolving gel buffer instead of the proper electrode buffer]]

	=== Visualizing Gels ===		=== Visualizing Gels ===

Janet B. Matsen: /* Prepping Gels */

2013-01-04T21:52:43Z

Prepping Gels

←Older revision		Revision as of 21:52, 4 January 2013
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	== '''Mistakes to Be Careful About''' ==		== '''Mistakes to Be Careful About''' ==
	=== Prepping Gels ===		=== Prepping Gels ===
-	* using an "old" APS solution when making gels. The 10% weight/volume APS ~~should~~ be made fresh each time for best results. Don't use a solution that is more than a month or so old. The gels won't polymerize.	+	* using an "old" APS solution when making gels. The 10% weight/volume APS can be made fresh each time for best results. Don't use a solution that is more than a month or so old. The gels won't polymerize.
	* not mixing the liquid gel mixture enough		* not mixing the liquid gel mixture enough
	** Janet pipettes up and down with a 10 or 15 mL pipette about 10 times, being careful not to bubble the solution.		** Janet pipettes up and down with a 10 or 15 mL pipette about 10 times, being careful not to bubble the solution.

Janet B. Matsen: /* Running Gels */

2013-01-04T00:53:49Z

Running Gels

←Older revision		Revision as of 00:53, 4 January 2013
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	** entire sample will leak out		** entire sample will leak out

-	=== ~~Running Gels~~ ===	+	=== Preparing Samples ===
	* using too much beta-mercaptoethanol in your sample buffer		* using too much beta-mercaptoethanol in your sample buffer
	** should have < 1% beta-mercaptoethanol in the mix after you add sample buffer to the		** should have < 1% beta-mercaptoethanol in the mix after you add sample buffer to the
	** too much reduction of cysteines is bad: will alter structure and even cleave proteins.		** too much reduction of cysteines is bad: will alter structure and even cleave proteins.
-	* sample sloshing out of the well you are ~~using~~ into a neighboring well	+
		+	=== Running Gels ===
		+	* sample sloshing out of the well you are trying to load and falling into a neighboring well
		+	* Electrode buffer leaking out from between the two plates
		+	** Gel will run very unusually or stop running. Keep an eye out!
		+	** avoid by making '''sure''' the plates are well-seated
	* Using Tris-HCl instead of Tris in the electrode buffer		* Using Tris-HCl instead of Tris in the electrode buffer
	* not having the lid to the running unit on all the way. --> poor electrical contact & blurry bands		* not having the lid to the running unit on all the way. --> poor electrical contact & blurry bands
-	* running the gel without enough buffer between the gel plates	+
-	** will cause the gel to run terribly (unusable)	+
-	** avoid by making '''sure''' the plates are well-seated	+
	=== Visualizing Gels ===		=== Visualizing Gels ===
	* Gels tear easily when warm.		* Gels tear easily when warm.

Janet B. Matsen: /* Prepping Gels */

2013-01-03T23:03:31Z

Prepping Gels

←Older revision		Revision as of 23:03, 3 January 2013
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	=== Prepping Gels ===		=== Prepping Gels ===
	* using an "old" APS solution when making gels. The 10% weight/volume APS should be made fresh each time for best results. Don't use a solution that is more than a month or so old. The gels won't polymerize.		* using an "old" APS solution when making gels. The 10% weight/volume APS should be made fresh each time for best results. Don't use a solution that is more than a month or so old. The gels won't polymerize.
-	[[image:gel with 1 month old APS and one with fresh APS.jpg\|thumb\|center\|The gel on the left was poured with a 1-month old APS solution. It appeared to polymerize, but the probein samples looked terrible. The gel on the right was poured with a fresh APS solution, but all other reagents identical.]]
	* not mixing the liquid gel mixture enough		* not mixing the liquid gel mixture enough
	** Janet pipettes up and down with a 10 or 15 mL pipette about 10 times, being careful not to bubble the solution.		** Janet pipettes up and down with a 10 or 15 mL pipette about 10 times, being careful not to bubble the solution.

Janet B. Matsen: /* Gel Prep */

2013-01-03T23:02:37Z

Gel Prep

←Older revision		Revision as of 23:02, 3 January 2013
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	=='''Gel Prep'''==		=='''Gel Prep'''==
-	* Clean cover plate and thicker spacer plate 75 mM	+	* Clean cover plate and thicker spacer plate (75 mM gap)
-	** Soap and Water	+	** Soap and Water: scrub with gloved fingers. Look to see if all chunks are gone.
-	**~~Ethanol~~	+	** Rinse with water, then ethanol. Prop up vertically against green clamps for stands so they drain and dry.
-	**DI water	+
-	* Dry plates	+
	* Setup one spacer plate and one cover plate in each gel holder		* Setup one spacer plate and one cover plate in each gel holder
	** The cover plate goes on the side of the spacer plate with the spacers in order to create a small gap between the plates		** The cover plate goes on the side of the spacer plate with the spacers in order to create a small gap between the plates
		+	** Put on the black counter top to pinch closed. Don't put it on the absorbant mat, as this slignt unevenness will increase your gel leakage rate. Janet had ~33% of gels leak until she started doing it on the black counter without padding; now she has ~5-10% of poured gels leak.
	* Put the gel holder into the casting stand		* Put the gel holder into the casting stand
	* '''Make a fresh 10% (w/vol) APS solution'''. Don't use an "old" solution; the gel won't polymerize. 10% w/v = 0.1 g/mL.		* '''Make a fresh 10% (w/vol) APS solution'''. Don't use an "old" solution; the gel won't polymerize. 10% w/v = 0.1 g/mL.
-	** 1 month old ~~APS often leads~~ to ~~poor polymerization. An example gel is shown below~~.	+	** 1 month old seems to be safe, but up to 3 months may be ok.

	=== '''Pour the Resolving Gel'''===		=== '''Pour the Resolving Gel'''===
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	*[http://mach7.bluehill.com/proteinc/tutorial/sdspage.html Gel Mix Recipe]		*[http://mach7.bluehill.com/proteinc/tutorial/sdspage.html Gel Mix Recipe]
	** Don't add APS/TEMED until ready to pour		** Don't add APS/TEMED until ready to pour
-	** '''The APS solution should be < 1 month old.'''	+	** '''The APS solution should be < 1 month old.''' Up to 2 months might be ok, but we don't have great data for this.
	*** A 3 month old solution failed to cause polymerization & the liquid spilled out from between the plates when released from the stand. -[[User:Janet B. Matsen\|JM 10/2012]]		*** A 3 month old solution failed to cause polymerization & the liquid spilled out from between the plates when released from the stand. -[[User:Janet B. Matsen\|JM 10/2012]]
-	*** A 1 month old solution gave wonky gels (couldn't tell there was poor polymerization until after staining.) See pic below. -[[User:Janet B. Matsen\|JM 1/2013]]	+	*** The BioRad protocols insist you should make it fresh every time, but we never do if we have one made within the last 4 weeks
-	*** The BioRad protocols insist you should make it fresh every time, but we never do if we have one made within the last 3 weeks	+	* Use pipette to put gel mix into the gap between the plates. Fill to about the bottom of the green line that is below the short plate.
-	* Use pipette to put gel mix into the gap between the plates	+
	* Carefully layer 50%EtOH 50% ddH2O on top of the gel to prevent the top of the gel from drying out		* Carefully layer 50%EtOH 50% ddH2O on top of the gel to prevent the top of the gel from drying out
-	* Let dry for ~~an hour~~	+	** It is very possible to ruin the desired evenness of the resolving gel by squirting this mixture in too fast. Janet has done it!
		+	* Let dry for 40 minutes.
		+	* Rinse ethanol/water mix off or you will get bubbles in the gel.
		+	** Rinse the gel aggressively by flushing with tap water from the sink.
		+	** Rinse several times with ddH2O
		+	** Prop the gel against the casting stand on its side so the liquid drains to one edge. Remove this with paper towel.
	* Store at 4 deg C wrapped in a wet paper towel and saran wrap if you're not going to use it right away.		* Store at 4 deg C wrapped in a wet paper towel and saran wrap if you're not going to use it right away.
		+	** You can also store in the fridge after pouring the stacking gel.

	=== '''Pour the Stacking Gel'''===		=== '''Pour the Stacking Gel'''===

-	* ~~Replace~~ gel in gel holder	+	* Place gel in gel holder & ensure no water is sitting on top of the resolving gel.
-	* ~~Rinse off~~ ethanol/water or butanol ~~mix used~~ to ~~keep the top of the gel hydrated~~	+	** Remove liquid on top if you didn't already do so. Removal of ethanol/water or butanol is important to prevent bubbles.
-	** not rinsing will result in bubbles between the gel and the plate (bad)	+	** Dry surface of gel carefully with Kimwipe or paper towel
-	* Dry surface of gel carefully with Kimwipe or paper towel	+
	** It can be a little gooey		** It can be a little gooey
	* Mix components of the amounts in the Gel Mix link. Mix in the order listed.		* Mix components of the amounts in the Gel Mix link. Mix in the order listed.
	**[http://mach7.bluehill.com/proteinc/tutorial/sdspage.html Gel Mix Recipe]		**[http://mach7.bluehill.com/proteinc/tutorial/sdspage.html Gel Mix Recipe]
	** Don't add APS/TEMED until ready to pour		** Don't add APS/TEMED until ready to pour
-	* Use pipette to put gel mix into the gap between the plates	+	* Use pipette to put gel mix into the gap between the plates.
		+	** Filling to the top reduces the probability of trapping bubbles under the comb when you insert the comb.
	* Insert the comb being '''careful not to trap any bubbles'''		* Insert the comb being '''careful not to trap any bubbles'''
	** It is much easier to avoid bubbles if you fill the space with enough solution that it spills over the top as you put the comb in. It also helps to lower one side completely, then lower the other side down.		** It is much easier to avoid bubbles if you fill the space with enough solution that it spills over the top as you put the comb in. It also helps to lower one side completely, then lower the other side down.
	* Attach 2 1" wide binder clips to each gel to help hold the comb in while drying. One on either side of the casting stand clamp.		* Attach 2 1" wide binder clips to each gel to help hold the comb in while drying. One on either side of the casting stand clamp.
-	** ~~We have not determined whether this is necessary~~ -[[User:Janet_B._Matsen\|JM 12/~~13/2012~~]]	+	** This will help ensure the edge wells don't get ruined -[[User:Janet_B._Matsen\|JM 1/2013]]
-	** On 1/3/2012 [[User:Janet_B._Matsen\|JM]] didn't use binder clips and lost an edge well on two out of 6 gels, which usually doesn't happen. ~~I recommend using the binder clips.~~	+	** On 1/3/2012 [[User:Janet_B._Matsen\|JM]] didn't use binder clips and lost an edge well on two out of 6 gels, which usually doesn't happen.
-	* Leave for ~~1 hour~~ while polymerization occurs.	+	* Leave for 30 minutes while polymerization occurs.
-	** Manuals actually suggest shorter times. 30 min should suffice.	+

	=== '''Wrapping up & Storing'''===		=== '''Wrapping up & Storing'''===
	* Put binder clips away		* Put binder clips away
-	* Rinse everything off	+	* Rinse everything off
	** Rinse gel under water with comb in to wash away solution that spilled over.		** Rinse gel under water with comb in to wash away solution that spilled over.
	** Remove comb from gel carefully (don't want to slant the parts that stick up) & soak in soapy water		** Remove comb from gel carefully (don't want to slant the parts that stick up) & soak in soapy water