Griffin:Western Blot Optimization - Revision history

Korey Griffin at 19:43, 1 October 2012

Korey Griffin — Mon, 01 Oct 2012 19:43:42 GMT

←Older revision		Revision as of 19:43, 1 October 2012
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	[[Image: WBimage4.jpg\|thumb\|No signal on a prolonged exposure could be an issue with either the primary antibody OR protein expression level]]		[[Image: WBimage4.jpg\|thumb\|No signal on a prolonged exposure could be an issue with either the primary antibody OR protein expression level]]

-	[[Image: ~~WBimage12~~.jpg\|thumb\|Excessive signal from heavy chain IP/WB @50 kD produces 'reverse banding']]	+	[[Image: WBimage13.jpg\|thumb\|Excessive signal from heavy chain IP/WB @50 kD produces 'reverse banding']]

	Time and labor at the bench is extremely valuable. Below is a guide to interpret western blotting experiences in a purposeful way so that each experiment will make a difference. Watch out for futile cycling in work flow and focus energy in a meaningful way.		Time and labor at the bench is extremely valuable. Below is a guide to interpret western blotting experiences in a purposeful way so that each experiment will make a difference. Watch out for futile cycling in work flow and focus energy in a meaningful way.

Korey Griffin at 19:43, 1 October 2012

Korey Griffin — Mon, 01 Oct 2012 19:43:17 GMT

←Older revision		Revision as of 19:43, 1 October 2012
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	[[Image: WBimage4.jpg\|thumb\|No signal on a prolonged exposure could be an issue with either the primary antibody OR protein expression level]]		[[Image: WBimage4.jpg\|thumb\|No signal on a prolonged exposure could be an issue with either the primary antibody OR protein expression level]]
		+
		+	[[Image: WBimage12.jpg\|thumb\|Excessive signal from heavy chain IP/WB @50 kD produces 'reverse banding']]

	Time and labor at the bench is extremely valuable. Below is a guide to interpret western blotting experiences in a purposeful way so that each experiment will make a difference. Watch out for futile cycling in work flow and focus energy in a meaningful way.		Time and labor at the bench is extremely valuable. Below is a guide to interpret western blotting experiences in a purposeful way so that each experiment will make a difference. Watch out for futile cycling in work flow and focus energy in a meaningful way.

Korey Griffin at 21:55, 11 April 2012

Korey Griffin — Wed, 11 Apr 2012 21:55:00 GMT

←Older revision		Revision as of 21:55, 11 April 2012
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	Commercial antibodies have a wide range of specificity and sensitivity for the protein of interest. Efficient blocking of the primary and secondary antibodies can take place in a variety of different blocking agents that contain soluble proteins and non-ionic detergent. When developing a new antibody by western blot, it is important to test different blocking/incubation buffers for the best possible signal:noise ratio in the assay. No single blocking agent is ideal for every primary antibody, since each antibody-antigen pair has unique binding characteristics.		Commercial antibodies have a wide range of specificity and sensitivity for the protein of interest. Efficient blocking of the primary and secondary antibodies can take place in a variety of different blocking agents that contain soluble proteins and non-ionic detergent. When developing a new antibody by western blot, it is important to test different blocking/incubation buffers for the best possible signal:noise ratio in the assay. No single blocking agent is ideal for every primary antibody, since each antibody-antigen pair has unique binding characteristics.
		+
		+	==Molecular weight vs. actual protein migration==

	Western blotting is a technique that separates proteins based on size. In general, the smaller the protein, the faster it migrates through the gel. However, migration is also affected by other factors, so the actual band size observed may differ from that predicted. Common factors include:		Western blotting is a technique that separates proteins based on size. In general, the smaller the protein, the faster it migrates through the gel. However, migration is also affected by other factors, so the actual band size observed may differ from that predicted. Common factors include:
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	4. Relative charge - the composition of amino acids (charged vs non-charged)		4. Relative charge - the composition of amino acids (charged vs non-charged)

-	5. Multimers - e.g. ~~dimerisation~~ of a protein. This is usually prevented in reducing conditions, although strong interactions can result in the appearance of higher bands	+	5. Multimers - e.g. dimerization of a protein. This is usually prevented in reducing conditions, although strong interactions can result in the appearance of higher bands

	==Always run a + control and Secondary control==		==Always run a + control and Secondary control==

Korey Griffin at 21:53, 11 April 2012

Korey Griffin — Wed, 11 Apr 2012 21:53:37 GMT

←Older revision		Revision as of 21:53, 11 April 2012
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	Commercial antibodies have a wide range of specificity and sensitivity for the protein of interest. Efficient blocking of the primary and secondary antibodies can take place in a variety of different blocking agents that contain soluble proteins and non-ionic detergent. When developing a new antibody by western blot, it is important to test different blocking/incubation buffers for the best possible signal:noise ratio in the assay. No single blocking agent is ideal for every primary antibody, since each antibody-antigen pair has unique binding characteristics.		Commercial antibodies have a wide range of specificity and sensitivity for the protein of interest. Efficient blocking of the primary and secondary antibodies can take place in a variety of different blocking agents that contain soluble proteins and non-ionic detergent. When developing a new antibody by western blot, it is important to test different blocking/incubation buffers for the best possible signal:noise ratio in the assay. No single blocking agent is ideal for every primary antibody, since each antibody-antigen pair has unique binding characteristics.
		+
		+	Western blotting is a technique that separates proteins based on size. In general, the smaller the protein, the faster it migrates through the gel. However, migration is also affected by other factors, so the actual band size observed may differ from that predicted. Common factors include:
		+
		+	1. Post-translational modification - e.g. phosphorylation, glycosylation etc, which increases the size of the protein
		+
		+	2. Post-translation cleavage - e.g. many proteins are synthesized as pro-proteins and then cleaved to give the active form, e.g. pro-caspases
		+
		+	3. Splice variants - alternative splicing may create different sized proteins produced from the same gene
		+
		+	4. Relative charge - the composition of amino acids (charged vs non-charged)
		+
		+	5. Multimers - e.g. dimerisation of a protein. This is usually prevented in reducing conditions, although strong interactions can result in the appearance of higher bands

	==Always run a + control and Secondary control==		==Always run a + control and Secondary control==

Korey Griffin at 21:52, 11 April 2012

Korey Griffin — Wed, 11 Apr 2012 21:52:04 GMT

←Older revision		Revision as of 21:52, 11 April 2012
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	[[Image: WBimage4.jpg\|thumb\|No signal on a prolonged exposure could be an issue with either the primary antibody OR protein expression level]]		[[Image: WBimage4.jpg\|thumb\|No signal on a prolonged exposure could be an issue with either the primary antibody OR protein expression level]]

-	~~Your time~~ and labor at the bench is extremely valuable. Below is a guide ~~that will enable you~~ to interpret ~~your~~ western blotting ~~experience~~ in a purposeful way so that each experiment will make a difference. Watch out for futile cycling in ~~your~~ work and focus ~~your~~ energy in a meaningful way.	+	Time and labor at the bench is extremely valuable. Below is a guide to interpret western blotting experiences in a purposeful way so that each experiment will make a difference. Watch out for futile cycling in work flow and focus energy in a meaningful way.

-	There is an increasing number of available commercial antibodies from several vendors. Depending ~~on who you buy from~~, the level of quality control ~~the vendor performs~~ is highly variable and this leaves ~~you with the~~ responsibility to optimize ~~your own protocol~~ in an efficient manner. Because every antibody is different, each one requires a different blocking/~~incbuation~~ buffer in order to optimize the signal:noise ratio.	+	There is an increasing number of available commercial antibodies from several vendors. Depending the vendor, the level of quality control is highly variable and this leaves a responsibility to optimize protocols in an efficient manner. Because every antibody is different, each one requires a different blocking/incubation buffer in order to optimize the signal:noise ratio.

	There are common themes in the types of incubation buffers that tend to work well for blocking and incubation.		There are common themes in the types of incubation buffers that tend to work well for blocking and incubation.

-	Commercial antibodies have a wide range of specificity and sensitivity for the protein of interest. ~~Effecient~~ blocking of the primary and secondary antibodies can take place in a variety of different blocking agents that contain soluble proteins and ~~nonionic~~ detergent. When developing a new antibody by western blot, it is important to test ~~several~~ different blocking/incubation buffers for the best possible signal:noise ratio in the assay. No single blocking agent is ideal for every primary antibody, since each antibody-antigen pair has unique binding characteristics.	+	Commercial antibodies have a wide range of specificity and sensitivity for the protein of interest. Efficient blocking of the primary and secondary antibodies can take place in a variety of different blocking agents that contain soluble proteins and non-ionic detergent. When developing a new antibody by western blot, it is important to test different blocking/incubation buffers for the best possible signal:noise ratio in the assay. No single blocking agent is ideal for every primary antibody, since each antibody-antigen pair has unique binding characteristics.

	==Always run a + control and Secondary control==		==Always run a + control and Secondary control==

Korey Griffin at 21:49, 11 April 2012

Korey Griffin — Wed, 11 Apr 2012 21:49:36 GMT

←Older revision		Revision as of 21:49, 11 April 2012
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	Commercial antibodies have a wide range of specificity and sensitivity for the protein of interest. Effecient blocking of the primary and secondary antibodies can take place in a variety of different blocking agents that contain soluble proteins and nonionic detergent. When developing a new antibody by western blot, it is important to test several different blocking/incubation buffers for the best possible signal:noise ratio in the assay. No single blocking agent is ideal for every primary antibody, since each antibody-antigen pair has unique binding characteristics.		Commercial antibodies have a wide range of specificity and sensitivity for the protein of interest. Effecient blocking of the primary and secondary antibodies can take place in a variety of different blocking agents that contain soluble proteins and nonionic detergent. When developing a new antibody by western blot, it is important to test several different blocking/incubation buffers for the best possible signal:noise ratio in the assay. No single blocking agent is ideal for every primary antibody, since each antibody-antigen pair has unique binding characteristics.

-	==Always run ~~your~~ + control and Secondary control==	+	==Always run a + control and Secondary control==

	*Running a + control is helpful in all situations where the banding profile for a given gene may have variation between cell/tissue types. Running the + control will determine if your system protein levels are in question vs. antibody titer/quality.		*Running a + control is helpful in all situations where the banding profile for a given gene may have variation between cell/tissue types. Running the + control will determine if your system protein levels are in question vs. antibody titer/quality.

Maximilian Peters: typo

Maximilian Peters — Thu, 12 May 2011 07:13:00 GMT

typo

←Older revision		Revision as of 07:13, 12 May 2011
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	There are common themes in the types of incubation buffers that tend to work well for blocking and incubation.		There are common themes in the types of incubation buffers that tend to work well for blocking and incubation.

-	Commercial antibodies have a wide range of specificity and sensitivity for the protein of interest. Effecient blocking of the primary and secondary antibodies can take place in a variety of different blocking agents that contain soluble proteins and nonionic detergent. When developing a new antibody by western blot, it is important to test several different blocking/incubation buffers for the best possible signal:noise ratio in the assay. No single blocking agent is ideal for every primary ~~antiobody~~, since each antibody-antigen pair has unique binding characteristics.	+	Commercial antibodies have a wide range of specificity and sensitivity for the protein of interest. Effecient blocking of the primary and secondary antibodies can take place in a variety of different blocking agents that contain soluble proteins and nonionic detergent. When developing a new antibody by western blot, it is important to test several different blocking/incubation buffers for the best possible signal:noise ratio in the assay. No single blocking agent is ideal for every primary antibody, since each antibody-antigen pair has unique binding characteristics.

	==Always run your + control and Secondary control==		==Always run your + control and Secondary control==

Korey Griffin at 19:46, 11 November 2010

Korey Griffin — Thu, 11 Nov 2010 19:46:11 GMT

←Older revision		Revision as of 19:46, 11 November 2010
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-
	[[Image: WBimage10.jpg\|thumb\|Tubulin loading control Western blot is an important control experiment to know your reagents and protocol are in line and that equal protein amounts are loaded in each lane]]		[[Image: WBimage10.jpg\|thumb\|Tubulin loading control Western blot is an important control experiment to know your reagents and protocol are in line and that equal protein amounts are loaded in each lane]]

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	Commercial antibodies have a wide range of specificity and sensitivity for the protein of interest. Effecient blocking of the primary and secondary antibodies can take place in a variety of different blocking agents that contain soluble proteins and nonionic detergent. When developing a new antibody by western blot, it is important to test several different blocking/incubation buffers for the best possible signal:noise ratio in the assay. No single blocking agent is ideal for every primary antiobody, since each antibody-antigen pair has unique binding characteristics.		Commercial antibodies have a wide range of specificity and sensitivity for the protein of interest. Effecient blocking of the primary and secondary antibodies can take place in a variety of different blocking agents that contain soluble proteins and nonionic detergent. When developing a new antibody by western blot, it is important to test several different blocking/incubation buffers for the best possible signal:noise ratio in the assay. No single blocking agent is ideal for every primary antiobody, since each antibody-antigen pair has unique binding characteristics.
		+
		+	==Always run your + control and Secondary control==
		+
		+	*Running a + control is helpful in all situations where the banding profile for a given gene may have variation between cell/tissue types. Running the + control will determine if your system protein levels are in question vs. antibody titer/quality.
		+
		+	*Running a secondary control in parallel will ensure that any artifact bands are clearly understood be.

	==The importance of the blocking/antibody incubation buffer==		==The importance of the blocking/antibody incubation buffer==

Korey Griffin: /* Re-applying ECL reagent */

Korey Griffin — Fri, 05 Nov 2010 22:09:58 GMT

Re-applying ECL reagent

←Older revision		Revision as of 22:09, 5 November 2010
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	===Re-applying ECL reagent===		===Re-applying ECL reagent===

-	The HRP (horseradish peroxidase) is an enzyme that oxidizes the ECL substrate. This oxidation reaction with chemiluminescent substrates produces light. The HRP enzyme can remain active as long as it is kept 4C in 1X TBS buffer, no azide~~). ECL contains HRP substrate as well as "enhancing" chemicals (modified phenols)~~. The moment you expose your blot with the HRP-conjugated antibody to the ECL, the substrate begins to be used up but it doesn't destroy the HRP enzyme.	+	The HRP (horseradish peroxidase) is an enzyme that oxidizes the ECL/luminol (contains HRP substrate + enhancing chemicals (modified phenols)). This oxidation reaction with chemiluminescent substrates produces light. The HRP enzyme can remain active as long as it is kept 4C in 1X TBS buffer, no azide. The moment you expose your blot with the HRP-conjugated antibody to the ECL, the substrate begins to be used up but it doesn't destroy the HRP enzyme.

	If banding signal is excessive:		If banding signal is excessive:
-	* Place membrane in 1XTBS overnight at 4C.	+	* Place membrane in 1XTBS overnight at 4C. The following day, simply re run the ECL step.
-	* The following day, simply re run the ECL step. ~~ECL contains HRP substrate as well as "enhancing" chemicals (modified phenols).~~	+
	* Blots can be kept in 1X TBS for a 2-3 days 4C. If the signal is too weak, reprobe with the secondary antibody or protein A-HRP, then add ECL reagent.		* Blots can be kept in 1X TBS for a 2-3 days 4C. If the signal is too weak, reprobe with the secondary antibody or protein A-HRP, then add ECL reagent.

Korey Griffin: /* Excess signal; multiple bands/too much banding */

Korey Griffin — Fri, 05 Nov 2010 22:07:58 GMT

Excess signal; multiple bands/too much banding

←Older revision		Revision as of 22:07, 5 November 2010
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	*post-translational modifications including glycosylation, nitrosylation, phosphorylation, methylation, acetylation, ubiquitination,		*post-translational modifications including glycosylation, nitrosylation, phosphorylation, methylation, acetylation, ubiquitination,
	*ubiquitin-dependent protein degradation		*ubiquitin-dependent protein degradation
		+
		+	===Re-applying ECL reagent===
		+
		+	The HRP (horseradish peroxidase) is an enzyme that oxidizes the ECL substrate. This oxidation reaction with chemiluminescent substrates produces light. The HRP enzyme can remain active as long as it is kept 4C in 1X TBS buffer, no azide). ECL contains HRP substrate as well as "enhancing" chemicals (modified phenols). The moment you expose your blot with the HRP-conjugated antibody to the ECL, the substrate begins to be used up but it doesn't destroy the HRP enzyme.
		+
		+	If banding signal is excessive:
		+	* Place membrane in 1XTBS overnight at 4C.
		+	* The following day, simply re run the ECL step. ECL contains HRP substrate as well as "enhancing" chemicals (modified phenols).
		+	* Blots can be kept in 1X TBS for a 2-3 days 4C. If the signal is too weak, reprobe with the secondary antibody or protein A-HRP, then add ECL reagent.

	==Excess signal; dark exposure==		==Excess signal; dark exposure==