User:Dhea Patel/Notebook/Experimental Biological Chemistry Notebook/2012/09/18 - Revision history

Dhea Patel: /* Data */

Dhea Patel — Wed, 28 Nov 2012 19:10:49 GMT

Data

←Older revision		Revision as of 19:10, 28 November 2012
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	[[Image:20120912_Absorbance_of_HRP_Assay_with_AAP.jpg]]		[[Image:20120912_Absorbance_of_HRP_Assay_with_AAP.jpg]]
	*A picture of all the various assay mixtures after the reaction had taken place.		*A picture of all the various assay mixtures after the reaction had taken place.
-
-	graphs depicting absorbance over time of enzymatic activity of HRP in a fix concentration of AAP and varied concentrations of hydrogen peroxide and depicting absorbance over time of enzymatic activity of HRP in a fixed concentration of hydrogen peroxide and varied concentrations of AAP.

	[[Image:Fixed_AAP_Varied_H2O2.png]]		[[Image:Fixed_AAP_Varied_H2O2.png]]
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	[[Image:Fixed_H2O2_Varied_AAP.png]]		[[Image:Fixed_H2O2_Varied_AAP.png]]
	*The graph depicts the absorbance over time of enzymatic activity of HRP in a fixed concentration of hydrogen peroxide and varied concentrations of AAP.		*The graph depicts the absorbance over time of enzymatic activity of HRP in a fixed concentration of hydrogen peroxide and varied concentrations of AAP.
-	*When the H<sub>2</sub>O<sub>2</sub> was fixed at 1.7mM, the reactions within the AAP concentration range of 156.25μM - 1.25mM showed a steady increase in absorptivity. The faster reactions with more absorptivity were the samples containing a more diluted amount of AAP solution.	+	*When the H<sub>2</sub>O<sub>2</sub> was fixed at 1.7mM, the reactions within the AAP concentration range of 156.25μM - 1.25mM showed a steady increase in absorptivity. The faster reactions with more absorptivity were the samples containing a more diluted amount of AAP solution.

	==Notes==		==Notes==

Dhea Patel at 19:10, 28 November 2012

Dhea Patel — Wed, 28 Nov 2012 19:10:24 GMT

←Older revision		Revision as of 19:10, 28 November 2012
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	''Making HRP''		''Making HRP''
		+	*MW of HRP = 40000 g/mol
	*1mg of HRP was dissolved in 1mL H<sub>2</sub>O, creating 23μM HRP solution.		*1mg of HRP was dissolved in 1mL H<sub>2</sub>O, creating 23μM HRP solution.
	*The HRP was further diluted to 2.3μM by adding 1mL HRP solution to 9mL H<sub>2</sub>O.		*The HRP was further diluted to 2.3μM by adding 1mL HRP solution to 9mL H<sub>2</sub>O.
-	*'''[[User:Abigail E. Miller\|Abigail E. Miller]] 20:08, 7 October 2012 (EDT)''':MW of HRP?

	'''2. Setting up the spectrophotometer'''		'''2. Setting up the spectrophotometer'''
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	\| 18 mM\|\|156.25uM\|\|1.7mM\|\|2.3uM		\| 18 mM\|\|156.25uM\|\|1.7mM\|\|2.3uM
	\|}		\|}
		+	*The concentrations above are the concentrations of each solution before being added to the cuvette.

-	*'''[[User:Abigail E. Miller\|Abigail E. Miller]] 20:08, 7 October 2012 (EDT)''':are these final concentrations or concentrations of solutions mixed?
	==Data==		==Data==

-	* Final concentrations of stock solutions in total volume of 1.5 mL:	+	* Final concentrations of stock solutions in the cuvette for a total volume of 1.5 mL:
-	*'''[[User:Abigail E. Miller\|Abigail E. Miller]] 20:08, 7 October 2012 (EDT)''':do you mean final concentrations in the cuvette	+
	{\| {{table}}		{\| {{table}}
	\| align="center" style="background:#f0f0f0;"\|'''Stock Solution'''		\| align="center" style="background:#f0f0f0;"\|'''Stock Solution'''
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	*A picture of all the various assay mixtures after the reaction had taken place.		*A picture of all the various assay mixtures after the reaction had taken place.

-	*'''[[~~User~~:~~Abigail E~~. ~~Miller\|Abigail E. Miller~~]] ~~20:08~~, ~~7 October 2012 (EDT)''':graphs~~ of ~~reactions should~~ be ~~posted here~~. ~~cannot support conclusions without data. all data should~~ be ~~included~~ in ~~your notebook~~.	+	graphs depicting absorbance over time of enzymatic activity of HRP in a fix concentration of AAP and varied concentrations of hydrogen peroxide and depicting absorbance over time of enzymatic activity of HRP in a fixed concentration of hydrogen peroxide and varied concentrations of AAP.
		+
		+	[[Image:Fixed_AAP_Varied_H2O2.png]]
		+	*This graph depicts the absorbance over time of enzymatic activity of HRP in a fix concentration of AAP and varied concentrations of hydrogen peroxide.
		+	*When the AAP was fixed at 2.5mM, the reaction with a concentration of 1.7mM H<sub>2</sub>O<sub>2</sub> took over 300 seconds to be completed. The reaction with a concentration of 850μM H<sub>2</sub>O<sub>2</sub> took about 200 seconds to be completed, and the majority of the reaction took place in the UV-vis, so the change in absorptivity was tracked and measured through out the entire reaction. Reactions with a hydrogen peroxide concentration less that 850μM appeared to occur too quickly such that the change in absorptivity was unable to be effectively tracked by the UV-vis.

-	*Please refer to [[~~User~~:~~Melissa_Novy/Notebook/CHEM-571/2012/09/18\|Melissa's entry~~]] ~~for graphs depicting absorbance over time of enzymatic activity of HRP in a fix concentration of AAP and varied concentrations of hydrogen peroxide and depicting~~ absorbance over time of enzymatic activity of HRP in a fixed concentration of hydrogen peroxide and varied concentrations of AAP.	+	[[Image:Fixed_H2O2_Varied_AAP.png]]
-	**When the AAP was fixed at 2.5mM, the reaction with a concentration of 1.7mM H<sub>2</sub>O<sub>2</sub> took over 300 seconds to be completed. The reaction with a concentration of 850μM H<sub>2</sub>O<sub>2</sub> took about 200 seconds to be completed, and the majority of the reaction took place in the UV-vis, so the change in absorptivity was tracked and measured through out the entire reaction. Reactions with a hydrogen peroxide concentration less that 850μM appeared to occur too quickly such that the change in absorptivity was unable to be effectively tracked by the UV-vis.	+	*The graph depicts the absorbance over time of enzymatic activity of HRP in a fixed concentration of hydrogen peroxide and varied concentrations of AAP.
-	**When the H<sub>2</sub>O<sub>2</sub> was fixed at 1.7mM, the reactions within the AAP concentration range of 156.25μM - 1.25mM showed a steady increase in absorptivity. The faster reactions with more absorptivity were the samples containing a more diluted amount of AAP solution.	+	*When the H<sub>2</sub>O<sub>2</sub> was fixed at 1.7mM, the reactions within the AAP concentration range of 156.25μM - 1.25mM showed a steady increase in absorptivity. The faster reactions with more absorptivity were the samples containing a more diluted amount of AAP solution.

	==Notes==		==Notes==

Abigail E. Miller at 00:08, 8 October 2012

Abigail E. Miller — Mon, 08 Oct 2012 00:08:38 GMT

←Older revision		Revision as of 00:08, 8 October 2012
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	*1mg of HRP was dissolved in 1mL H<sub>2</sub>O, creating 23μM HRP solution.		*1mg of HRP was dissolved in 1mL H<sub>2</sub>O, creating 23μM HRP solution.
	*The HRP was further diluted to 2.3μM by adding 1mL HRP solution to 9mL H<sub>2</sub>O.		*The HRP was further diluted to 2.3μM by adding 1mL HRP solution to 9mL H<sub>2</sub>O.
		+	*'''[[User:Abigail E. Miller\|Abigail E. Miller]] 20:08, 7 October 2012 (EDT)''':MW of HRP?

	'''2. Setting up the spectrophotometer'''		'''2. Setting up the spectrophotometer'''
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	****The concentration of H<sub>2</sub>O<sub>2</sub> was varied by serial dilution as follows, while the concentrations of all other solutions were kept constant		****The concentration of H<sub>2</sub>O<sub>2</sub> was varied by serial dilution as follows, while the concentrations of all other solutions were kept constant
	*0.05mL HRP was added to each cuvette at the last moment, right before the cuvette was placed in the UV-vis.		*0.05mL HRP was added to each cuvette at the last moment, right before the cuvette was placed in the UV-vis.
-


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	\|}		\|}

		+	*'''[[User:Abigail E. Miller\|Abigail E. Miller]] 20:08, 7 October 2012 (EDT)''':are these final concentrations or concentrations of solutions mixed?
	==Data==		==Data==

	* Final concentrations of stock solutions in total volume of 1.5 mL:		* Final concentrations of stock solutions in total volume of 1.5 mL:
		+	*'''[[User:Abigail E. Miller\|Abigail E. Miller]] 20:08, 7 October 2012 (EDT)''':do you mean final concentrations in the cuvette
	{\| {{table}}		{\| {{table}}
	\| align="center" style="background:#f0f0f0;"\|'''Stock Solution'''		\| align="center" style="background:#f0f0f0;"\|'''Stock Solution'''
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	*A picture of all the various assay mixtures after the reaction had taken place.		*A picture of all the various assay mixtures after the reaction had taken place.

		+	*'''[[User:Abigail E. Miller\|Abigail E. Miller]] 20:08, 7 October 2012 (EDT)''':graphs of reactions should be posted here. cannot support conclusions without data. all data should be included in your notebook.

	*Please refer to [[User:Melissa_Novy/Notebook/CHEM-571/2012/09/18\|Melissa's entry]] for graphs depicting absorbance over time of enzymatic activity of HRP in a fix concentration of AAP and varied concentrations of hydrogen peroxide and depicting absorbance over time of enzymatic activity of HRP in a fixed concentration of hydrogen peroxide and varied concentrations of AAP.		*Please refer to [[User:Melissa_Novy/Notebook/CHEM-571/2012/09/18\|Melissa's entry]] for graphs depicting absorbance over time of enzymatic activity of HRP in a fix concentration of AAP and varied concentrations of hydrogen peroxide and depicting absorbance over time of enzymatic activity of HRP in a fixed concentration of hydrogen peroxide and varied concentrations of AAP.

Dhea Patel: /* Description */

Dhea Patel — Tue, 02 Oct 2012 18:36:02 GMT

Description

←Older revision		Revision as of 18:36, 2 October 2012
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	''Making HRP''		''Making HRP''
	*1mg of HRP was dissolved in 1mL H<sub>2</sub>O, creating 23μM HRP solution.		*1mg of HRP was dissolved in 1mL H<sub>2</sub>O, creating 23μM HRP solution.
-	*The HRP was further diluted to 2.~~3μMmM~~ by adding 1mL HRP solution to 9mL H<sub>2</sub>O.	+	*The HRP was further diluted to 2.3μM by adding 1mL HRP solution to 9mL H<sub>2</sub>O.

	'''2. Setting up the spectrophotometer'''		'''2. Setting up the spectrophotometer'''

Dhea Patel at 18:24, 2 October 2012

Dhea Patel — Tue, 02 Oct 2012 18:24:20 GMT

←Older revision		Revision as of 18:24, 2 October 2012
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	*Please refer to [[User:Melissa_Novy/Notebook/CHEM-571/2012/09/18\|Melissa's entry]] for graphs depicting absorbance over time of enzymatic activity of HRP in a fix concentration of AAP and varied concentrations of hydrogen peroxide and depicting absorbance over time of enzymatic activity of HRP in a fixed concentration of hydrogen peroxide and varied concentrations of AAP.		*Please refer to [[User:Melissa_Novy/Notebook/CHEM-571/2012/09/18\|Melissa's entry]] for graphs depicting absorbance over time of enzymatic activity of HRP in a fix concentration of AAP and varied concentrations of hydrogen peroxide and depicting absorbance over time of enzymatic activity of HRP in a fixed concentration of hydrogen peroxide and varied concentrations of AAP.
		+	**When the AAP was fixed at 2.5mM, the reaction with a concentration of 1.7mM H<sub>2</sub>O<sub>2</sub> took over 300 seconds to be completed. The reaction with a concentration of 850μM H<sub>2</sub>O<sub>2</sub> took about 200 seconds to be completed, and the majority of the reaction took place in the UV-vis, so the change in absorptivity was tracked and measured through out the entire reaction. Reactions with a hydrogen peroxide concentration less that 850μM appeared to occur too quickly such that the change in absorptivity was unable to be effectively tracked by the UV-vis.
		+	**When the H<sub>2</sub>O<sub>2</sub> was fixed at 1.7mM, the reactions within the AAP concentration range of 156.25μM - 1.25mM showed a steady increase in absorptivity. The faster reactions with more absorptivity were the samples containing a more diluted amount of AAP solution.
		+
		+	==Notes==
		+	The protocol that this experiment was based off of was found in the DropBox folder, "CHEM571_Fall2012" → "HRPAssays" → "3760-Protocol HRP_AbsorbanceAssay.pdf"

	<!-- ##### DO NOT edit below this line unless you know what you are doing. ##### -->		<!-- ##### DO NOT edit below this line unless you know what you are doing. ##### -->

Dhea Patel: /* Description */

Dhea Patel — Tue, 02 Oct 2012 18:11:09 GMT

Description

←Older revision		Revision as of 18:11, 2 October 2012
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	''Making AAP Solution''		''Making AAP Solution''
	*0.125g of AAP was added to 5mL H<sub>2</sub>O to make 0.0025M solution of AAP.		*0.125g of AAP was added to 5mL H<sub>2</sub>O to make 0.0025M solution of AAP.
-	*'''Making Phenol solution'''	+	*''Making Phenol solution''
	**0.4054g 4-iodophenol was added to 20mL H<sub>2</sub>O to make 0.17M phenol.		**0.4054g 4-iodophenol was added to 20mL H<sub>2</sub>O to make 0.17M phenol.
	***Because the phenol was not dissolving in the water, 700μM DMSO was added to the mixture.		***Because the phenol was not dissolving in the water, 700μM DMSO was added to the mixture.

Dhea Patel: /* Objective */

Dhea Patel — Tue, 02 Oct 2012 18:07:54 GMT

Objective

←Older revision		Revision as of 18:07, 2 October 2012
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	==Objective==		==Objective==

-	#to make stock solutions of sodium phosphate, 4-aminoantipyrine (AAP), H<sub>2</sub>O<sub>2</sub>, and horseradish peroxidase (HRP)	+	*to make stock solutions of sodium phosphate, 4-aminoantipyrine (AAP), H<sub>2</sub>O<sub>2</sub>, and horseradish peroxidase (HRP)
-	#to perform an assay on the enzymatic rate of HRP and observing the kinetics of the reaction via real-time UV-vis.	+	*to perform an assay on the enzymatic rate of HRP and observing the kinetics of the reaction via real-time UV-vis.
-	##Vary the concentration of ~~H2O2~~, while keeping the concentration of AAP constant, then vary the concentration of APP while keeping the concentration of ~~H2O2~~ constant.	+	**Vary the concentration of H<sub>2</sub>O<sub>2</sub>, while keeping the concentration of AAP constant, then vary the concentration of APP while keeping the concentration of H<sub>2</sub>O<sub>2</sub> constant.

	==Description==		==Description==

Dhea Patel: /* Data */

Dhea Patel — Wed, 26 Sep 2012 18:59:55 GMT

Data

←Older revision		Revision as of 18:59, 26 September 2012
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	\|}		\|}

-	*[[Image:20120912_Absorbance_of_HRP_Assay_with_AAP.jpg]]	+
-	A picture of all the various assay mixtures after the reaction had taken place.	+	[[Image:20120912_Absorbance_of_HRP_Assay_with_AAP.jpg]]
		+	*A picture of all the various assay mixtures after the reaction had taken place.
		+

	*Please refer to [[User:Melissa_Novy/Notebook/CHEM-571/2012/09/18\|Melissa's entry]] for graphs depicting absorbance over time of enzymatic activity of HRP in a fix concentration of AAP and varied concentrations of hydrogen peroxide and depicting absorbance over time of enzymatic activity of HRP in a fixed concentration of hydrogen peroxide and varied concentrations of AAP.		*Please refer to [[User:Melissa_Novy/Notebook/CHEM-571/2012/09/18\|Melissa's entry]] for graphs depicting absorbance over time of enzymatic activity of HRP in a fix concentration of AAP and varied concentrations of hydrogen peroxide and depicting absorbance over time of enzymatic activity of HRP in a fixed concentration of hydrogen peroxide and varied concentrations of AAP.

Dhea Patel at 18:59, 26 September 2012

Dhea Patel — Wed, 26 Sep 2012 18:59:36 GMT

←Older revision		Revision as of 18:59, 26 September 2012
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	\|-		\|-
	\|}		\|}
		+
		+	*[[Image:20120912_Absorbance_of_HRP_Assay_with_AAP.jpg]]
		+	A picture of all the various assay mixtures after the reaction had taken place.

	*Please refer to [[User:Melissa_Novy/Notebook/CHEM-571/2012/09/18\|Melissa's entry]] for graphs depicting absorbance over time of enzymatic activity of HRP in a fix concentration of AAP and varied concentrations of hydrogen peroxide and depicting absorbance over time of enzymatic activity of HRP in a fixed concentration of hydrogen peroxide and varied concentrations of AAP.		*Please refer to [[User:Melissa_Novy/Notebook/CHEM-571/2012/09/18\|Melissa's entry]] for graphs depicting absorbance over time of enzymatic activity of HRP in a fix concentration of AAP and varied concentrations of hydrogen peroxide and depicting absorbance over time of enzymatic activity of HRP in a fixed concentration of hydrogen peroxide and varied concentrations of AAP.

Dhea Patel: /* Data */

Dhea Patel — Tue, 25 Sep 2012 17:38:29 GMT

Data

←Older revision		Revision as of 17:38, 25 September 2012
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	\|}		\|}

-	*Please refer to [[User:Melissa_Novy/Notebook/CHEM-571/2012/09/18\|Melissa's entry] for graphs depicting absorbance over time of enzymatic activity of HRP in a fix concentration of AAP and varied concentrations of hydrogen peroxide and depicting absorbance over time of enzymatic activity of HRP in a fixed concentration of hydrogen peroxide and varied concentrations of AAP.	+	*Please refer to [[User:Melissa_Novy/Notebook/CHEM-571/2012/09/18\|Melissa's entry]] for graphs depicting absorbance over time of enzymatic activity of HRP in a fix concentration of AAP and varied concentrations of hydrogen peroxide and depicting absorbance over time of enzymatic activity of HRP in a fixed concentration of hydrogen peroxide and varied concentrations of AAP.

	<!-- ##### DO NOT edit below this line unless you know what you are doing. ##### -->		<!-- ##### DO NOT edit below this line unless you know what you are doing. ##### -->