Immunoprecipitation - Revision history

Davebrid: added categories

2016-08-21T14:23:03Z

added categories

← Older revision		Revision as of 14:23, 21 August 2016
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	* for tissue lysates - use 0.5 mg of protein, add primary antibody first for 0.5 hour and then add beads.		* for tissue lysates - use 0.5 mg of protein, add primary antibody first for 0.5 hour and then add beads.
	* rotate 1 hour to overnight in cold room		* rotate 1 hour to overnight in cold room
	* spin 2 minutes at 5000 rpm		* spin 2 minutes at 5000 rpm and aspirate supernatent, carefully avoiding beads (can use crushed tip).

	and aspirate supernatent, carefully avoiding beads (can use crushed tip).
	* wash X3: add 1 ml lysis buffer, mix by inverting tube (no vortex), spin 2 minutes 5000 rpm and aspirate supernatent.		* wash X3: add 1 ml lysis buffer, mix by inverting tube (no vortex), spin 2 minutes 5000 rpm and aspirate supernatent.
	* after last wash and aspiration, spin once more and aspirate all supernatant.		* after last wash and aspiration, spin once more and aspirate all supernatant.
	* add 50 ul of sample buffer, boil 5 minutes.		* add 50 ul of sample buffer, boil 5 minutes.
	* to get rid of beads - open cap (very important!!!), prick bottom with 25G needle up to halfway of bevel (can use eppendorf tube holder~~, as in picture [[File:Needle_in_eppendorf_2.jpg\|100px]]~~). Place tube on new eppendorf tube, close cap and see drop flowing through the tiny hole. Spin with hinges of both tubes towards center of centrifuge, without centrifuge lid, for 30 seconds at 4600 rpm.		* to get rid of beads - open cap (very important!!!), prick bottom with 25G needle up to halfway of bevel (can use eppendorf tube holder). Place tube on new eppendorf tube, close cap and see drop flowing through the tiny hole. Spin with hinges of both tubes towards center of centrifuge, without centrifuge lid, for 30 seconds at 4600 rpm.
	* load 10ul of IP on gel.		* load 10ul of IP on gel.
	* load original lysate - 1% of original IP lysate volume loaded (when using 0.5ml and loading 10/50ul of final IP, this will be 2ul of lysate 1:1 with sample buffer).		* load original lysate - 1% of original IP lysate volume loaded (when using 0.5ml and loading 10/50ul of final IP, this will be 2ul of lysate 1:1 with sample buffer).

			[[ Category: Immunoprecipitation‏‎ ]]
			[[ Category: Protein ]]
			[[ Category: Protein-Protein Interactions ]]

Irith at 19:33, 3 July 2012

2012-07-03T19:33:40Z

← Older revision		Revision as of 19:33, 3 July 2012
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	* scrape cells in lysis buffer, such as [[RIPA Buffer]] with protease inhibitors and phosphatase inhibitors		* scrape cells in lysis buffer, such as [[RIPA Buffer]] with protease inhibitors and phosphatase inhibitors
	* for each well in a 12 well plate lyse in 1 ml.		* for each well in a 12 well plate lyse in 1 ml.
	* combine 0.5 ml lysate, 2 ul of antibody and 25 ul of protein A/G plus agarose beads (catalog # sc 2003 santa cruz).		* combine 0.5 ml lysate, 0.2-0.5 ug of antibody and 25 ul of protein A/G plus agarose beads (catalog # sc 2003 santa cruz).
			* for tissue lysates - use 0.5 mg of protein, add primary antibody first for 0.5 hour and then add beads.
	* rotate 1 hour to overnight in cold room		* rotate 1 hour to overnight in cold room
	* spin 2 minutes at 5000 rpm and aspirate supernatent, carefully avoiding beads (can use crushed tip).		* spin 2 minutes at 5000 rpm

			and aspirate supernatent, carefully avoiding beads (can use crushed tip).
	* wash X3: add 1 ml lysis buffer, mix by inverting tube (no vortex), spin 2 minutes 5000 rpm and aspirate supernatent.		* wash X3: add 1 ml lysis buffer, mix by inverting tube (no vortex), spin 2 minutes 5000 rpm and aspirate supernatent.
	* after last wash and aspiration, spin once more and aspirate all supernatant.		* after last wash and aspiration, spin once more and aspirate all supernatant.

Irith at 15:42, 5 April 2012

2012-04-05T15:42:06Z

← Older revision		Revision as of 15:42, 5 April 2012
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	==Protocol==		==Protocol==

	~~samples~~ are on ice/in cold		Samples are always kept on ice/in cold room/4 celsius centrifuge
	* ~~Scrape~~ cells in lysis buffer, such as RIPA ~~buffer~~ with protease inhibitors and phosphatase inhibitors ~~(see [[RIPA Buffer]]).~~		* wash cells once with PBS
	* ~~For~~ each well in a 12 well plate lyse in 1 ml.		* scrape cells in lysis buffer, such as [[RIPA Buffer]] with protease inhibitors and phosphatase inhibitors
			* for each well in a 12 well plate lyse in 1 ml.
	* combine 0.5 ml lysate, 2 ul of antibody and 25 ul of protein A/G plus agarose beads (catalog # sc 2003 santa cruz).		* combine 0.5 ml lysate, 2 ul of antibody and 25 ul of protein A/G plus agarose beads (catalog # sc 2003 santa cruz).
	* rotate 1 hour-overnight in cold room		* rotate 1 hour to overnight in cold room
	* spin 2 minutes at 5000 rpm and aspirate supernatent, carefully avoiding beads		* spin 2 minutes at 5000 rpm and aspirate supernatent, carefully avoiding beads (can use crushed tip).
	* wash X3: add 1 ml lysis buffer, mix by inverting tube, spin and aspirate supernatent ~~as before~~.		* wash X3: add 1 ml lysis buffer, mix by inverting tube (no vortex), spin 2 minutes 5000 rpm and aspirate supernatent.
	* spin once more and aspirate all supernatant.		* after last wash and aspiration, spin once more and aspirate all supernatant.
	* add 50 ul of sample buffer, boil 5 minutes		* add 50 ul of sample buffer, boil 5 minutes.
	* to get rid of beads - open cap, prick bottom with 25G needle [[File:Needle_in_eppendorf_2.jpg\|100px]] (~~up to halfway~~ of ~~bevel~~).		* to get rid of beads - open cap (very important!!!), prick bottom with 25G needle up to halfway of bevel (can use eppendorf tube holder, as in picture [[File:Needle_in_eppendorf_2.jpg\|100px]]). Place tube on new eppendorf tube, close cap and see drop flowing through the tiny hole. Spin with hinges of both tubes towards center of centrifuge, without centrifuge lid, for 30 seconds at 4600 rpm.
			* load 10ul of IP on gel.
			* load original lysate - 1% of original IP lysate volume loaded (when using 0.5ml and loading 10/50ul of final IP, this will be 2ul of lysate 1:1 with sample buffer).

Irith at 15:29, 5 April 2012

2012-04-05T15:29:21Z

← Older revision		Revision as of 15:29, 5 April 2012
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	* spin once more and aspirate all supernatant.		* spin once more and aspirate all supernatant.
	* add 50 ul of sample buffer, boil 5 minutes		* add 50 ul of sample buffer, boil 5 minutes
	* to get rid of beads - open cap, prick bottom with 25G needle [[File:Needle_in_eppendorf_2.jpg\|~~50px~~]] (up to halfway of bevel).		* to get rid of beads - open cap, prick bottom with 25G needle [[File:Needle_in_eppendorf_2.jpg\|100px]] (up to halfway of bevel).

Irith at 15:29, 5 April 2012

2012-04-05T15:29:07Z

← Older revision		Revision as of 15:29, 5 April 2012
Line 10:		Line 10:
	* spin once more and aspirate all supernatant.		* spin once more and aspirate all supernatant.
	* add 50 ul of sample buffer, boil 5 minutes		* add 50 ul of sample buffer, boil 5 minutes
	* to get rid of beads - open cap, prick bottom with 25G needle [[File:Needle_in_eppendorf_2.jpg\|~~300px~~]] (up to halfway of bevel).		* to get rid of beads - open cap, prick bottom with 25G needle [[File:Needle_in_eppendorf_2.jpg\|50px]] (up to halfway of bevel).

Irith at 15:28, 5 April 2012

2012-04-05T15:28:49Z

← Older revision		Revision as of 15:28, 5 April 2012
Line 10:		Line 10:
	* spin once more and aspirate all supernatant.		* spin once more and aspirate all supernatant.
	* add 50 ul of sample buffer, boil 5 minutes		* add 50 ul of sample buffer, boil 5 minutes
	* to get rid of beads - open cap, prick bottom with ~~27G~~ needle (up to halfway of bevel). ~~can~~		* to get rid of beads - open cap, prick bottom with 25G needle [[File:Needle_in_eppendorf_2.jpg\|300px]] (up to halfway of bevel).

Irith at 15:26, 5 April 2012

2012-04-05T15:26:05Z

← Older revision		Revision as of 15:26, 5 April 2012
Line 1:		Line 1:
	==Protocol==		==Protocol==
	* ~~Lyse~~ cells in lysis buffer, such as RIPA buffer with protease inhibitors and phosphatase inhibitors (see [[RIPA Buffer]]).
			samples are on ice/in cold
			* Scrape cells in lysis buffer, such as RIPA buffer with protease inhibitors and phosphatase inhibitors (see [[RIPA Buffer]]).
	* For each well in a 12 well plate lyse in 1 ml.		* For each well in a 12 well plate lyse in 1 ml.
	* ~~rotate~~ 0.5 ml lysate ~~with~~ 25 ul of protein A/G plus agarose beads (catalog # sc 2003 santa cruz).		* combine 0.5 ml lysate, 2 ul of antibody and 25 ul of protein A/G plus agarose beads (catalog # sc 2003 santa cruz).
			* rotate 1 hour-overnight in cold room
			* spin 2 minutes at 5000 rpm and aspirate supernatent, carefully avoiding beads
			* wash X3: add 1 ml lysis buffer, mix by inverting tube, spin and aspirate supernatent as before.
			* spin once more and aspirate all supernatant.
			* add 50 ul of sample buffer, boil 5 minutes
			* to get rid of beads - open cap, prick bottom with 27G needle (up to halfway of bevel). can

Davebrid: updated formatting

2012-04-04T17:12:53Z

updated formatting

← Older revision		Revision as of 17:12, 4 April 2012
Line 1:		Line 1:
	Lyse cells in lysis buffer, such as RIPA buffer with protease inhibitors and phosphatase inhibitors (see ~~link~~).		==Protocol==
	For each well in a 12 well plate lyse in 1 ml.		* Lyse cells in lysis buffer, such as RIPA buffer with protease inhibitors and phosphatase inhibitors (see [[RIPA Buffer]]).
	rotate 0.5 ml lysate with 25 ul of protein A/G plus agarose beads (catalog # sc 2003 santa cruz).		* For each well in a 12 well plate lyse in 1 ml.
			* rotate 0.5 ml lysate with 25 ul of protein A/G plus agarose beads (catalog # sc 2003 santa cruz).

Irith: Created page with 'Lyse cells in lysis buffer, such as RIPA buffer with protease inhibitors and phosphatase inhibitors (see link). For each well in a 12 well plate lyse in 1 ml. rotate 0.5 ml lysat...'

2012-04-04T16:42:15Z

Created page with 'Lyse cells in lysis buffer, such as RIPA buffer with protease inhibitors and phosphatase inhibitors (see link). For each well in a 12 well plate lyse in 1 ml. rotate 0.5 ml lysat...'

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Lyse cells in lysis buffer, such as RIPA buffer with protease inhibitors and phosphatase inhibitors (see link).
For each well in a 12 well plate lyse in 1 ml.
rotate 0.5 ml lysate with 25 ul of protein A/G plus agarose beads (catalog # sc 2003 santa cruz).