Free Sulfhydryl Determination - Revision history

Christopher C Vanlang: /* Reagents */

Christopher C Vanlang — Mon, 25 Jun 2012 21:55:57 GMT

Reagents

←Older revision		Revision as of 21:55, 25 June 2012
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	==Reagents==		==Reagents==

-	*'DTNB Solution' 20x	+	*'''DTNB Solution''' 20x
	**50 mM NaAc		**50 mM NaAc
	**2mM DTNB		**2mM DTNB
-	*'Tris Buffer Solution' 10x	+	*'''Tris Buffer Solution''' 10x
	**1M Tris pH 8.0		**1M Tris pH 8.0
-	*Thiol Standard	+	*'''Thiol Standard'''
	**100 mM DTT		**100 mM DTT
	*Protein of Interest		*Protein of Interest
-		+	**Usually several proteins samples are compared: a stock solution, a reduced and buffer exchanged sample, a reduced sample that was reacted with a thiol-blocking compound (like iodoacetate)and buffer exchanged. Make sure to remove free thiol or thiol-reactive compounds from the protein solution before attempting to measure free cysteine concentration.
-	~~===Protein of Interest (not in a thiol-containing buffer!)===~~	+
-	Usually several proteins samples are compared: a stock solution, a reduced and buffer exchanged sample, a reduced sample that was reacted with a thiol-blocking compound (like iodoacetate)and buffer exchanged. Make sure to remove free thiol or thiol-reactive compounds from the protein solution before attempting to measure free cysteine concentration.	+
-		+

	==Protocol==		==Protocol==

Christopher C Vanlang at 21:54, 25 June 2012

Christopher C Vanlang — Mon, 25 Jun 2012 21:54:58 GMT

←Older revision		Revision as of 21:54, 25 June 2012
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	==Reagents==		==Reagents==

-	~~===1 M~~ Tris~~-Cl, pH 8.0===~~	+	*'DTNB Solution' 20x
-	~~tris base to~~ pH 8.0 ~~with HCl~~	+	**50 mM NaAc
		+	**2mM DTNB
		+	*'Tris Buffer Solution' 10x
		+	**1M Tris pH 8.0
		+	*Thiol Standard
		+	**100 mM DTT
		+	*Protein of Interest

-	~~===2 mM DTNB dissolved in 50 mM sodium acetate===~~
-	~~===Stock of known thiol compound (I use 100 mM DTT)===~~
-	~~DTT has 2 reactive thiols per molecule, this will be corrected for later~~
	===Protein of Interest (not in a thiol-containing buffer!)===		===Protein of Interest (not in a thiol-containing buffer!)===
	Usually several proteins samples are compared: a stock solution, a reduced and buffer exchanged sample, a reduced sample that was reacted with a thiol-blocking compound (like iodoacetate)and buffer exchanged. Make sure to remove free thiol or thiol-reactive compounds from the protein solution before attempting to measure free cysteine concentration.		Usually several proteins samples are compared: a stock solution, a reduced and buffer exchanged sample, a reduced sample that was reacted with a thiol-blocking compound (like iodoacetate)and buffer exchanged. Make sure to remove free thiol or thiol-reactive compounds from the protein solution before attempting to measure free cysteine concentration.
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	==Protocol==		==Protocol==

-	Prepare a "<b>Working Solution</b>" by mixing:	+	#Prepare a "<b>Working Solution</b>" by mixing:
		+	#*8.4 mLs Water
		+	#*1 mL 1M Tris-Cl pH 8.0
		+	#*0.5 mL DTNB solution
		+	#Make serial dilutions of DTT (or whatever control you are using)with the highest concentration about 5 mM (or 10 mM for single thiol compound).
		+	#Aliquot <b>Working Solution</b> into tubes (495 μL is usually fine).
		+	#Add 5 μL of the DTT solutions to each and mix.
		+	#Measure the absorbance of the solution at 412 nm (blank against a no DTT mixture). If you have trouble with reproducibility of the same sample, try placing the cuvette with the solution in the spec., then blanking, and without removing the cuvette, adding the thiol, mixing and measuring.
		+	#Multiply the measured 412 absorbance by 100 (you made a 1/100 dilution in the reaction).
		+	#Divide the result by 2 if your compound had 2 reactive groups (like DTT).
		+	#Divide the result by 13,600 (the extinction coefficient of the yellow guy at 412 nm)
		+	##This is the molarity of the yellow reagent that arose from a reaction with the free thiols in the solution you tested. You can plot this against the concentration of DTT you thought you used to make sure the values are close.
		+	#In my hands, ther reaction is linear thoughout the linear range of the spec. (~0.01-1.0).

-	*8.4 mLs Water	+	==96 well Plate Assay Protocol==
		+	#Prepare a "<b>Working Solution</b>" by mixing:
		+	#*8.4 mLs Water
		+	#*1 mL 1M Tris-Cl pH 8.0
		+	#*0.5 mL DTNB solution
		+	#Add 99 μL working solution into plate for each
		+	#Add 1 μL of sample to each and mix
		+	#Allow the reaction to take place for 5 minutes
		+	#Measure 412 absorbance

-	*1 mL 1M Tris-Cl pH 8.0
-
-	*0.5 mL DTNB solution
-
-	~~Make serial dilutions of DTT (or whatever control you are using)with the highest concentration about 5 mM (or 10 mM for single thiol compound).~~
-
-	~~Aliquot <b>Working Solution</b> into tubes (495 μL is usually fine).~~
-
-	~~Add 5 μL of the DTT solutions to each and mix.~~
-
-	Measure the absorbance of the solution at 412 nm (blank against a no DTT mixture). If you have trouble with reproducibility of the same sample, try placing the cuvette with the solution in the spec., then blanking, and without removing the cuvette, adding the thiol, mixing and measuring.
-
-	~~Multiply the measured 412 absorbance by 100 (you made a 1/100 dilution in the reaction).~~
-
-	~~Divide the result by 2 if your compound had 2 reactive groups (like DTT).~~
-
-	~~Divide the result by 13,600 (the extinction coefficient of the yellow guy at 412 nm)~~
-
-	This is the molarity of the yellow reagent that arose from a reaction with the free thiols in the solution you tested. You can plot this against the concentration of DTT you thought you used to make sure the values are close.
-
-	~~In my hands, ther reaction is linear thoughout the linear range of the spec. (~0.01-1.0).~~

	==Comments==		==Comments==

Reshma P. Shetty at 00:34, 9 May 2007

Reshma P. Shetty — Wed, 09 May 2007 00:34:48 GMT

←Older revision		Revision as of 00:34, 9 May 2007
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	[[Category: In vitro]]		[[Category: In vitro]]
	[[Category: Protein]]		[[Category: Protein]]
-	~~[[Category: Free Sulfhydryl Determination]]~~

Jenny T Nguyen at 20:53, 13 July 2006

Jenny T Nguyen — Thu, 13 Jul 2006 20:53:28 GMT

←Older revision		Revision as of 20:53, 13 July 2006
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	2) Bulaj, G.; Kortemme, T.; Goldenberg, D. P. (1998) Biochemistry 37, 8965-8972. (Recent usage)		2) Bulaj, G.; Kortemme, T.; Goldenberg, D. P. (1998) Biochemistry 37, 8965-8972. (Recent usage)
		+
		+	[[Category: Protocol]]
		+	[[Category: In vitro]]
		+	[[Category: Protein]]
		+	[[Category: Free Sulfhydryl Determination]]

Smoore: /* Protocol */

Smoore — Thu, 02 Mar 2006 19:52:18 GMT

Protocol

←Older revision		Revision as of 19:52, 2 March 2006
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	Make serial dilutions of DTT (or whatever control you are using)with the highest concentration about 5 mM (or 10 mM for single thiol compound).		Make serial dilutions of DTT (or whatever control you are using)with the highest concentration about 5 mM (or 10 mM for single thiol compound).

-	Aliquot <b>Working Solution</b> into tubes (495 uL is usually fine).	+	Aliquot <b>Working Solution</b> into tubes (495 μL is usually fine).

-	Add 5 uL of the DTT solutions to each and mix.	+	Add 5 μL of the DTT solutions to each and mix.

	Measure the absorbance of the solution at 412 nm (blank against a no DTT mixture). If you have trouble with reproducibility of the same sample, try placing the cuvette with the solution in the spec., then blanking, and without removing the cuvette, adding the thiol, mixing and measuring.		Measure the absorbance of the solution at 412 nm (blank against a no DTT mixture). If you have trouble with reproducibility of the same sample, try placing the cuvette with the solution in the spec., then blanking, and without removing the cuvette, adding the thiol, mixing and measuring.

Smoore at 19:08, 2 March 2006

Smoore — Thu, 02 Mar 2006 19:08:46 GMT

←Older revision		Revision as of 19:08, 2 March 2006
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		+	Submitted by [[Sean Moore]]
		+
	==Background==		==Background==
	This is the protocol I used to determine the concentration of reduced cysteine in a purified protein. It takes advantage of the redox potential of the sulhydryl group and a coliometric reagent that turns yellow upon reaction with the sulhydryl (DTNB + SH ---> 2-nitro-5-thiobenzoic acid (yellow)).		This is the protocol I used to determine the concentration of reduced cysteine in a purified protein. It takes advantage of the redox potential of the sulhydryl group and a coliometric reagent that turns yellow upon reaction with the sulhydryl (DTNB + SH ---> 2-nitro-5-thiobenzoic acid (yellow)).
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	==Comments==		==Comments==
		+
		+	References
		+
		+	1) Ellman, G. L. (1959) Arch. Biochem. Biophys. 82, 70-77. (Original determination)
		+
		+	2) Bulaj, G.; Kortemme, T.; Goldenberg, D. P. (1998) Biochemistry 37, 8965-8972. (Recent usage)

Smoore at 19:06, 2 March 2006

Smoore — Thu, 02 Mar 2006 19:06:28 GMT

←Older revision		Revision as of 19:06, 2 March 2006
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	===Protein of Interest (not in a thiol-containing buffer!)===		===Protein of Interest (not in a thiol-containing buffer!)===
	Usually several proteins samples are compared: a stock solution, a reduced and buffer exchanged sample, a reduced sample that was reacted with a thiol-blocking compound (like iodoacetate)and buffer exchanged. Make sure to remove free thiol or thiol-reactive compounds from the protein solution before attempting to measure free cysteine concentration.		Usually several proteins samples are compared: a stock solution, a reduced and buffer exchanged sample, a reduced sample that was reacted with a thiol-blocking compound (like iodoacetate)and buffer exchanged. Make sure to remove free thiol or thiol-reactive compounds from the protein solution before attempting to measure free cysteine concentration.
		+

	==Protocol==		==Protocol==
-	Prepare a "Working Solution" by mixing:	+
		+	Prepare a "<b>Working Solution</b>" by mixing:

	*8.4 mLs Water		*8.4 mLs Water
Line 30:		Line 32:
	Add 5 uL of the DTT solutions to each and mix.		Add 5 uL of the DTT solutions to each and mix.

-	Measure the absorbance of the solution at 412 nm (blank against a no DTT mixture). If you have trouble with reproducibility of the same sample, try placing the cuvette with the solution in the spec., then blanking, and without removing the cuvette, adding the thiol and measuring.	+	Measure the absorbance of the solution at 412 nm (blank against a no DTT mixture). If you have trouble with reproducibility of the same sample, try placing the cuvette with the solution in the spec., then blanking, and without removing the cuvette, adding the thiol, mixing and measuring.
		+
		+	Multiply the measured 412 absorbance by 100 (you made a 1/100 dilution in the reaction).
		+
		+	Divide the result by 2 if your compound had 2 reactive groups (like DTT).
		+
		+	Divide the result by 13,600 (the extinction coefficient of the yellow guy at 412 nm)
		+
		+	This is the molarity of the yellow reagent that arose from a reaction with the free thiols in the solution you tested. You can plot this against the concentration of DTT you thought you used to make sure the values are close.
		+
		+	In my hands, ther reaction is linear thoughout the linear range of the spec. (~0.01-1.0).

	==Comments==		==Comments==

Smoore: /* Protocol */

Smoore — Thu, 02 Mar 2006 18:00:26 GMT

Protocol

←Older revision		Revision as of 18:00, 2 March 2006
Line 28:		Line 28:
	Aliquot <b>Working Solution</b> into tubes (495 uL is usually fine).		Aliquot <b>Working Solution</b> into tubes (495 uL is usually fine).

-	Add 5 uL of the DTT solutions to each	+	Add 5 uL of the DTT solutions to each and mix.
		+
		+	Measure the absorbance of the solution at 412 nm (blank against a no DTT mixture). If you have trouble with reproducibility of the same sample, try placing the cuvette with the solution in the spec., then blanking, and without removing the cuvette, adding the thiol and measuring.

	==Comments==		==Comments==

Smoore: /* Protocol */

Smoore — Thu, 02 Mar 2006 17:58:03 GMT

Protocol

←Older revision		Revision as of 17:58, 2 March 2006
Line 16:		Line 16:

	==Protocol==		==Protocol==
		+	Prepare a "Working Solution" by mixing:
		+
		+	*8.4 mLs Water
		+
		+	*1 mL 1M Tris-Cl pH 8.0
		+
		+	*0.5 mL DTNB solution
		+
		+	Make serial dilutions of DTT (or whatever control you are using)with the highest concentration about 5 mM (or 10 mM for single thiol compound).
		+
		+	Aliquot <b>Working Solution</b> into tubes (495 uL is usually fine).
		+
		+	Add 5 uL of the DTT solutions to each

	==Comments==		==Comments==

Smoore: /* Protein of Interest (not in a thiol-containing buffer!) */

Smoore — Thu, 02 Mar 2006 15:47:31 GMT

Protein of Interest (not in a thiol-containing buffer!)

←Older revision		Revision as of 15:47, 2 March 2006
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	DTT has 2 reactive thiols per molecule, this will be corrected for later		DTT has 2 reactive thiols per molecule, this will be corrected for later
	===Protein of Interest (not in a thiol-containing buffer!)===		===Protein of Interest (not in a thiol-containing buffer!)===
-	Usually several proteins samples are ~~compare~~: stock solution, reduced and buffer exchanged, reduced ~~and~~ reacted with a thiol-blocking compound (like iodoacetate)and buffer exchanged. Make sure to remove ~~whatever~~ free thiol or thiol-reactive compounds from the protein solution before attempting to measure free cysteine concentration.	+	Usually several proteins samples are compared: a stock solution, a reduced and buffer exchanged sample, a reduced sample that was reacted with a thiol-blocking compound (like iodoacetate)and buffer exchanged. Make sure to remove free thiol or thiol-reactive compounds from the protein solution before attempting to measure free cysteine concentration.

	==Protocol==		==Protocol==

	==Comments==		==Comments==