Griffin: Ultimate Immunoprecipitation Guide - Revision history

Korey Griffin at 20:50, 17 April 2012

Korey Griffin — Tue, 17 Apr 2012 20:50:11 GMT

←Older revision		Revision as of 20:50, 17 April 2012
Line 4:		Line 4:


-	~~If you are planning on~~ performing an IP experiment, then consider to run a preliminary western blot first in order to get a feel for what the antibody is capable of binding.	+	When performing an IP experiment, then consider to run a preliminary western blot first in order to get a feel for what the antibody is capable of binding.

-	RIPA ('''R'''adio '''I'''mmuno'''P'''reciptation '''A'''ssay) buffer is a traditional name for an array of recipes that have found success over the years. Below are details that can contribute to optimizing ~~your~~ immunoprecipitation ~~experiment~~. Lysis buffer components can and will influence the efficiency of the IP reaction. Detergent composition is a major factor for IP reactions. Adjusting salt concentration and detergent composition will influence the efficiency of the IP reaction. Membrane bound proteins (proteins based in lipid rafts), protein complexes, and protein charge can all influence the efficient yield of ~~your~~ gene product in the lysate prep.	+	RIPA ('''R'''adio '''I'''mmuno'''P'''reciptation '''A'''ssay) buffer is a traditional name for an array of recipes that have found success over the years. Below are details that can contribute to optimizing immunoprecipitation experiments. Lysis buffer components can and will influence the efficiency of the IP reaction. Detergent composition is a major factor for IP reactions. Adjusting salt concentration and detergent composition will influence the efficiency of the IP reaction. Membrane bound proteins (proteins based in lipid rafts), protein complexes, and protein charge can all influence the efficient yield of a gene product isolate within the lysate prep.

	*The amount of protein present in cells will vary with cell type. 1 to 10 million cells can yield ~1mg of protein.		*The amount of protein present in cells will vary with cell type. 1 to 10 million cells can yield ~1mg of protein.

Korey Griffin at 20:48, 17 April 2012

Korey Griffin — Tue, 17 Apr 2012 20:48:44 GMT

←Older revision		Revision as of 20:48, 17 April 2012
Line 13:		Line 13:

	==Common RIPA components==		==Common RIPA components==
-	~~[[Image:Protein AGL Site Binding to IgG.jpg\|thumb\|right\|Protein A, G, L binding to IgG]]~~

	PBS : Salt prevents non-specific protein aggregation		PBS : Salt prevents non-specific protein aggregation
Line 80:		Line 79:

	[[Image:antibodybinding.jpg\|thumb\|right\|Binding Affinity Guide]]		[[Image:antibodybinding.jpg\|thumb\|right\|Binding Affinity Guide]]
		+
		+	[[Image:Protein AGL Site Binding to IgG.jpg\|thumb\|right\|Protein A, G, L binding to IgG]]

	Protein A & Protein G bind to most mammalian immunoglobulins primarily through their Fc regions. Protein L is a kappa light chain specific Ig-binding protein.		Protein A & Protein G bind to most mammalian immunoglobulins primarily through their Fc regions. Protein L is a kappa light chain specific Ig-binding protein.

Korey Griffin at 20:47, 17 April 2012

Korey Griffin — Tue, 17 Apr 2012 20:47:32 GMT

←Older revision		Revision as of 20:47, 17 April 2012
Line 13:		Line 13:

	==Common RIPA components==		==Common RIPA components==
		+	[[Image:Protein AGL Site Binding to IgG.jpg\|thumb\|right\|Protein A, G, L binding to IgG]]

	PBS : Salt prevents non-specific protein aggregation		PBS : Salt prevents non-specific protein aggregation

Korey Griffin: /* Common RIPA components */

Korey Griffin — Thu, 05 Jan 2012 21:31:46 GMT

Common RIPA components

←Older revision		Revision as of 21:31, 5 January 2012
Line 27:		Line 27:

	0.1% SDS : Ionic detergent to extract membrane protein and isolate lipids		0.1% SDS : Ionic detergent to extract membrane protein and isolate lipids
		+
		+	PMSF : Stock Solution 100 mM (100x stock) or 200 mM in Isopropanol; use @ 1 mM. Store PMSF solution up to 6 months @ 4C.

	EGTA : Protease Inhibitor, Prevents protein degradation. You can make your own, or several vendors have convenient crushable pills that form a protease inhibitor cocktail solution.		EGTA : Protease Inhibitor, Prevents protein degradation. You can make your own, or several vendors have convenient crushable pills that form a protease inhibitor cocktail solution.

Korey Griffin: /* Common RIPA components */

Korey Griffin — Thu, 20 Oct 2011 17:21:45 GMT

Common RIPA components

←Older revision		Revision as of 17:21, 20 October 2011
Line 34:		Line 34:
	NaF (Sodium Fluoride) : Serine/Threonine phosphatase Inhibitor; hydrostatic interference of active sights of phosphatases		NaF (Sodium Fluoride) : Serine/Threonine phosphatase Inhibitor; hydrostatic interference of active sights of phosphatases

-	~~Misc.~~ Phosphatase Inhibitors: Phosphorylation/dephosphorylation of proteins influences ~~the charge-charge~~ relationships ~~that proteins have with eachother in solution~~. Proteins undergo covalent attachment of a phosphoryl group (phosphorylation) ~~typically~~ at serine, threonine, or tyrosine residues. Phosphate groups are ~~removeable~~ via protein phosphatases. During the extraction of phosphorylated proteins from cell and tissue, preserving the phosphorylation states of total protein ~~is a good technique~~.	+	===Phosphatase Inhibitors===
		+
		+	Phosphorylation/dephosphorylation of proteins influences hydrostatic relationships. Proteins undergo covalent attachment of a phosphoryl group (phosphorylation) at serine, threonine, or tyrosine residues. Phosphate groups are removable via protein phosphatases. During the extraction of phosphorylated proteins from cell and tissue, there are advantages to preserving the phosphorylation states of total protein.
		+
		+	*(-)-p-Bromotetramisole oxalate
		+	*Cantharidin
		+	*Calyculin A
		+	*Discodermia calyx
		+	*Imidazole
		+	*Sodium Fluoride
		+	*Sodium Molybdate
		+	*Sodium Orthovanadate
		+	*Sodium Tartrate Dihydrate

	===Protease Inhibitor Cocktails===		===Protease Inhibitor Cocktails===

Korey Griffin: /* Protease Inhibitor Cocktails */

Korey Griffin — Tue, 17 May 2011 22:51:35 GMT

Protease Inhibitor Cocktails

←Older revision		Revision as of 22:51, 17 May 2011
Line 46:		Line 46:
	*04693116001: Complete™ Roche Protease Inhibitor Cocktail Tablets		*04693116001: Complete™ Roche Protease Inhibitor Cocktail Tablets
	*sc-29131: Santa Cruz Biotechnology Inc.,Protease Inhibitor Cocktail Tablet, EDTA-free		*sc-29131: Santa Cruz Biotechnology Inc.,Protease Inhibitor Cocktail Tablet, EDTA-free
		+
		+	====Protease Inhibitor components====
		+
		+	*Aprotinin
		+	*Bestatin
		+	*Calpain Inhibitor I & II
		+	*Chymostatin
		+	*E-64
		+	*Leupeptin
		+	*Alfa-2 Macroglobulin
		+	*Perfabloc SC
		+	*Pepstatin
		+	*PMSF
		+	*TLCK-HCl
		+	*Trypsin Inhibitor (chicken egg white, soybean)

	==Protein A/G/L Agarose==		==Protein A/G/L Agarose==

Korey Griffin at 00:48, 19 March 2011

Korey Griffin — Sat, 19 Mar 2011 00:48:58 GMT

←Older revision		Revision as of 00:48, 19 March 2011
Line 9:		Line 9:

	*The amount of protein present in cells will vary with cell type. 1 to 10 million cells can yield ~1mg of protein.		*The amount of protein present in cells will vary with cell type. 1 to 10 million cells can yield ~1mg of protein.
-		+	*70% confluent 10 cm dish can yield ~600-1000 ug total protein
	*Certain types of proteins (ie insoluble, modified) may escape classical lysis and purification procedures under the mild conditions outlined below. Additional methods that may improve on enrichment include cross-linking and purification under denaturing conditions.		*Certain types of proteins (ie insoluble, modified) may escape classical lysis and purification procedures under the mild conditions outlined below. Additional methods that may improve on enrichment include cross-linking and purification under denaturing conditions.

Korey Griffin at 00:46, 19 March 2011

Korey Griffin — Sat, 19 Mar 2011 00:46:36 GMT

←Older revision		Revision as of 00:46, 19 March 2011
Line 1:		Line 1:
	[[Image:humanisotypetable.jpg\|thumb\|right\|Serum levels of human Ig isotypes]]		[[Image:humanisotypetable.jpg\|thumb\|right\|Serum levels of human Ig isotypes]]
		+	[[Image:proteingbindingcapacity.jpg\|thumb\|right\|Protein G Binding Capacity]]
		+	[[Image:proteinabindingcapacity.jpg\|thumb\|right\|Protein A Binding Capacity]]
		+

	If you are planning on performing an IP experiment, then consider to run a preliminary western blot first in order to get a feel for what the antibody is capable of binding.		If you are planning on performing an IP experiment, then consider to run a preliminary western blot first in order to get a feel for what the antibody is capable of binding.

Korey Griffin at 16:30, 19 July 2010

Korey Griffin — Mon, 19 Jul 2010 16:30:17 GMT

←Older revision		Revision as of 16:30, 19 July 2010
Line 5:		Line 5:
	RIPA ('''R'''adio '''I'''mmuno'''P'''reciptation '''A'''ssay) buffer is a traditional name for an array of recipes that have found success over the years. Below are details that can contribute to optimizing your immunoprecipitation experiment. Lysis buffer components can and will influence the efficiency of the IP reaction. Detergent composition is a major factor for IP reactions. Adjusting salt concentration and detergent composition will influence the efficiency of the IP reaction. Membrane bound proteins (proteins based in lipid rafts), protein complexes, and protein charge can all influence the efficient yield of your gene product in the lysate prep.		RIPA ('''R'''adio '''I'''mmuno'''P'''reciptation '''A'''ssay) buffer is a traditional name for an array of recipes that have found success over the years. Below are details that can contribute to optimizing your immunoprecipitation experiment. Lysis buffer components can and will influence the efficiency of the IP reaction. Detergent composition is a major factor for IP reactions. Adjusting salt concentration and detergent composition will influence the efficiency of the IP reaction. Membrane bound proteins (proteins based in lipid rafts), protein complexes, and protein charge can all influence the efficient yield of your gene product in the lysate prep.

-	The amount of protein present in cells will vary with cell type. 1 to 10 million cells can yield ~1mg of protein.	+	*The amount of protein present in cells will vary with cell type. 1 to 10 million cells can yield ~1mg of protein.

-	Certain types of proteins (ie insoluble, modified) may escape classical lysis and purification procedures under the mild conditions outlined below. Additional methods that may improve on enrichment include cross-linking and purification under denaturing conditions.	+	*Certain types of proteins (ie insoluble, modified) may escape classical lysis and purification procedures under the mild conditions outlined below. Additional methods that may improve on enrichment include cross-linking and purification under denaturing conditions.

	==Common RIPA components==		==Common RIPA components==

Korey Griffin at 16:29, 19 July 2010

Korey Griffin — Mon, 19 Jul 2010 16:29:57 GMT

←Older revision		Revision as of 16:29, 19 July 2010
Line 6:		Line 6:

	The amount of protein present in cells will vary with cell type. 1 to 10 million cells can yield ~1mg of protein.		The amount of protein present in cells will vary with cell type. 1 to 10 million cells can yield ~1mg of protein.
		+
		+	Certain types of proteins (ie insoluble, modified) may escape classical lysis and purification procedures under the mild conditions outlined below. Additional methods that may improve on enrichment include cross-linking and purification under denaturing conditions.

	==Common RIPA components==		==Common RIPA components==