User:Melissa Novy/Notebook/CHEM-571/2012/09/26 - Revision history

Melissa Novy at 16:42, 18 October 2012

2012-10-18T16:42:13Z

←Older revision		Revision as of 16:42, 18 October 2012
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	* Continue optimizing the HRP chemiluminescence assay by adjusting the concentrations of the reactants.		* Continue optimizing the HRP chemiluminescence assay by adjusting the concentrations of the reactants.
	* Analyze Tris buffers at pH 8 and 10 and at various concentrations with UV-vis spectroscopy.		* Analyze Tris buffers at pH 8 and 10 and at various concentrations with UV-vis spectroscopy.
-	*~~'''~~[[User:~~Abigail E. Miller~~\|~~Abigail~~ E. ~~Miller~~]] 18:~~14, 7 October~~ 2012 ~~(EDT)''~~'~~:didn't you also need ot do something with~~ the ~~cells~~ for ~~making~~ the ~~ADA protein?~~	+
		+	==Cell Protein Expression==
		+	* Protocol
		+	*# The four inoculated starter cultures, made on [[User:Melissa_Novy/Notebook/CHEM-571/2012/09/25\|2012/09/25]] were centrifuged in a Sorvali RC6+ centrifuge for 15 min at 4°C and at 4500 rpm.
		+	*# The supernatant was discarded and the four resultant <i>E. coli</i> cell pellets were resuspended in 4 mL each of luria broth from the expression cultures, made on [[User:Melissa_Novy/Notebook/CHEM-571/2012/09/25\|2012/09/25]].
		+	*# The resuspended solutions were added to the expression cultures and the flasks were incubated and shaken at 37°C and 160 rpm.
		+	*# 1 mL of 0.4 M IPTG was added to each of the flasks. Please refer to [[User:Dhea_Patel/Notebook/Experimental_Biological_Chemistry_Notebook/2012/09/26\|Dhea Patel's entry]] for how the IPTG solution was made.
		+	*# Incubation and shaking of the flasks was resumed for 3 hours.
		+	*# The solutions were centrifuged at 4500 rpm and at 4°C for a period of 15 min to pellet the cells.
		+	*# The supernatant was discarded and the cell pellets were resuspended in 30 mL of binding buffer, made today, to give a total of four suspended cell solutions.
		+	*# The solutions were stored at -80°C.

	==Binding and Elution Buffers==		==Binding and Elution Buffers==
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	[[Image:Tris_Buffer_Absorbance.JPG]]		[[Image:Tris_Buffer_Absorbance.JPG]]
	* Graph of absorbance versus wavelength of Tris buffer in H<sub>2</sub>O at pH 8 and 10 and at concentrations ranging from 10 μM to 10 mM.		* Graph of absorbance versus wavelength of Tris buffer in H<sub>2</sub>O at pH 8 and 10 and at concentrations ranging from 10 μM to 10 mM.
		+	** The Tris buffers were made on [[User:Melissa_Novy/Notebook/CHEM-571/2012/09/04\|2012/09/04]].
	* Note that very small peaks were observed around 730-740 nm for all solutions and another small, broad peak was observed around 600 nm for the 10 μM Tris buffer solution at pH 8. The data indicate that absorbance of Tris buffer does not affect the peak observed for AuNPs at around 540 nm and the concentration and pH of Tris does not significantly affect the peaks for Tris observed.		* Note that very small peaks were observed around 730-740 nm for all solutions and another small, broad peak was observed around 600 nm for the 10 μM Tris buffer solution at pH 8. The data indicate that absorbance of Tris buffer does not affect the peak observed for AuNPs at around 540 nm and the concentration and pH of Tris does not significantly affect the peaks for Tris observed.
-
-
-	*'''[[User:Abigail E. Miller\|Abigail E. Miller]] 18:11, 7 October 2012 (EDT)''':it is not at all clear that this is tris buffer added to AuNPs. and there is no link to where the AuNPs came from or when these solutions were mixed. or is that not what this is about?

	==HRP Chemiluminescence Assay==		==HRP Chemiluminescence Assay==

Melissa Novy: /* HRP Chemiluminescence Assay */

2012-10-18T16:28:26Z

HRP Chemiluminescence Assay

←Older revision		Revision as of 16:28, 18 October 2012
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	* It was observed that the fluorescence spectra obtained from HRP-luminol assay solutions were producing negative intensity values. To determine why this was the case, a blank of carbonate buffer was also analyzed (see graph). As this blank also produced negative intensity values, it was determined that the baseline was negative and some fluorescence was actually being produced by the assay solutions.		* It was observed that the fluorescence spectra obtained from HRP-luminol assay solutions were producing negative intensity values. To determine why this was the case, a blank of carbonate buffer was also analyzed (see graph). As this blank also produced negative intensity values, it was determined that the baseline was negative and some fluorescence was actually being produced by the assay solutions.
	* The HRP concentration was further varied from trials on [[User:Melissa_Novy/Notebook/CHEM-571/2012/09/25\|2012/09/25]] in an attempt to slow the reaction rate such that the fluorimeter could detect the decline in fluorescence.		* The HRP concentration was further varied from trials on [[User:Melissa_Novy/Notebook/CHEM-571/2012/09/25\|2012/09/25]] in an attempt to slow the reaction rate such that the fluorimeter could detect the decline in fluorescence.
-	* ~~???~~	+	* Suggestions to produce better results are to alter the pH and to add H<sub>2</sub>O<sub>2</sub> to begin the reaction instead of HRP.

	==Notes==		==Notes==

Melissa Novy at 16:15, 17 October 2012

2012-10-17T16:15:10Z

←Older revision		Revision as of 16:15, 17 October 2012
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	** Make binding buffer and elution buffer.		** Make binding buffer and elution buffer.
	** Please refer to [[User:Dhea_Patel/Notebook/Experimental_Biological_Chemistry_Notebook/2012/09/26\|Dhea Patel's entry]] for the protocols and calculations of the above solutions.		** Please refer to [[User:Dhea_Patel/Notebook/Experimental_Biological_Chemistry_Notebook/2012/09/26\|Dhea Patel's entry]] for the protocols and calculations of the above solutions.
-	*'''[[User:Abigail E. Miller\|Abigail E. Miller]] 18:09, 7 October 2012 (EDT)''':you need to include the specifics of the binding and elution buffers, pH final volume and concentration of each component.
	* Continue optimizing the HRP chemiluminescence assay by adjusting the concentrations of the reactants.		* Continue optimizing the HRP chemiluminescence assay by adjusting the concentrations of the reactants.
	* Analyze Tris buffers at pH 8 and 10 and at various concentrations with UV-vis spectroscopy.		* Analyze Tris buffers at pH 8 and 10 and at various concentrations with UV-vis spectroscopy.
	*'''[[User:Abigail E. Miller\|Abigail E. Miller]] 18:14, 7 October 2012 (EDT)''':didn't you also need ot do something with the cells for making the ADA protein?		*'''[[User:Abigail E. Miller\|Abigail E. Miller]] 18:14, 7 October 2012 (EDT)''':didn't you also need ot do something with the cells for making the ADA protein?
		+
		+	==Binding and Elution Buffers==
		+	* Note that these buffers will be used to purify the proteins extracted from <i>E. coli</i> to ultimately obtain adenosine deaminase.
		+	* After the buffers were made, the pH of each was adjusted to 7.5 by adding 1 N HCl dropwise and monitoring the pH with a Thermo Scientific Orion 5 Star pH meter.
		+	* 1 L of binding buffer was made with components of imidazole, Tris, and NaCl at the following concentrations in 1 L of water:
		+	** 0.03 M imidazole
		+	** 0.02 M Tris
		+	** 0.5 M NaCl
		+	* 500 mL of elution buffer was made with components of imidazole, Tris, and NaCl at the following concentrations in 500 mL of water:
		+	** 0.5 M imidazole
		+	** 0.02 M Tris
		+	** 0.5 M NaCl

	==Spectroscopy Data of Tris Buffers==		==Spectroscopy Data of Tris Buffers==

Melissa Novy: /* Notes */

2012-10-09T15:40:03Z

Notes

←Older revision		Revision as of 15:40, 9 October 2012
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	\| HRP\|\|66		\| HRP\|\|66
	\|}		\|}
		+	* These volumes were used to calculate the final concentrations of the solution components in the assay solutions.
		+
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	__NOTOC__		__NOTOC__

Melissa Novy: /* HRP Chemiluminescence Assay */

2012-10-09T15:38:36Z

HRP Chemiluminescence Assay

←Older revision		Revision as of 15:38, 9 October 2012
Line 33:		Line 33:

	==HRP Chemiluminescence Assay==		==HRP Chemiluminescence Assay==
		+	* Solutions made on [[User:Melissa_Novy/Notebook/CHEM-571/2012/09/25\|2012/09/25]] were used to continue the HRP-luminol assay.
	* Concentrations of solutions tested:		* Concentrations of solutions tested:
	{\| {{table}}		{\| {{table}}
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	\|-		\|-
	\| 4\|\|18 mM\|\|1.25 mM\|\|1.7 mM\|\|4.6 µM		\| 4\|\|18 mM\|\|1.25 mM\|\|1.7 mM\|\|4.6 µM
		+	\|}
		+	<br>
		+	* Initial and final concentrations (in a total volume of ~2 mL) of all HRP-luminol reaction solutions that gave usable results:
		+	{\| {{table}}
		+	\| align="center" style="background:#f0f0f0;"\|''''''
		+	\| align="center" style="background:#f0f0f0;"\|'''Initial Concentrations [mM]'''
		+	\| align="center" style="background:#f0f0f0;"\|''''''
		+	\| align="center" style="background:#f0f0f0;"\|''''''
		+	\| align="center" style="background:#f0f0f0;"\|''''''
		+	\| align="center" style="background:#f0f0f0;"\|'''Final Concentrations [mM]'''
		+	\| align="center" style="background:#f0f0f0;"\|''''''
		+	\| align="center" style="background:#f0f0f0;"\|''''''
		+	\| align="center" style="background:#f0f0f0;"\|''''''
		+	\|-
		+	\| Trial\|\|4-Iodophenol\|\|Luminol\|\|H<sub>2</sub>O<sub>2</sub>\|\|HRP\|\|4-Iodophenol\|\|Luminol\|\|H<sub>2</sub>O<sub>2</sub>\|\|HRP
		+	\|-
		+	\| 1\|\|18\|\|1.25\|\|1.7\|\|0.0023\|\|0.000119\|\|0.000578\|\|0.000846\|\|7.63E-08
		+	\|-
		+	\| 2\|\|18\|\|0.625\|\|1.7\|\|0.0023\|\|0.000119\|\|0.000289\|\|0.000846\|\|7.63E-08
		+	\|-
		+	\| 3\|\|18\|\|0.625\|\|0.85\|\|0.0023\|\|0.000119\|\|0.000289\|\|0.000423\|\|7.63E-08
		+	\|-
		+	\| 4\|\|18\|\|1.25\|\|0.425\|\|0.0023\|\|0.000119\|\|0.000578\|\|0.000212\|\|7.63E-08
		+	\|-
		+	\| 5\|\|18\|\|1.25\|\|1.7\|\|0.0023\|\|0.000119\|\|0.000578\|\|0.000846\|\|7.63E-08
		+	\|-
		+	\| 6\|\|18\|\|1.25\|\|1.7\|\|0.00115\|\|0.000119\|\|0.000578\|\|0.000846\|\|3.82E-08
	\|}		\|}
	<br>		<br>
	[[Image:HRP_Chemiluminescence_Assay.JPG]]		[[Image:HRP_Chemiluminescence_Assay.JPG]]
	* Graph of intensity versus time of 4-iodophenol-enhanced solutions of hydrogen peroxide, luminol, and HRP.		* Graph of intensity versus time of 4-iodophenol-enhanced solutions of hydrogen peroxide, luminol, and HRP.
-	* ~~Refer to~~ the ~~[[Media: HRP_Assays~~.~~pdf\|report]] for analysis~~ of ~~HRP assays with AAP~~ and ~~luminol~~.	+	* It was observed that the fluorescence spectra obtained from HRP-luminol assay solutions were producing negative intensity values. To determine why this was the case, a blank of carbonate buffer was also analyzed (see graph). As this blank also produced negative intensity values, it was determined that the baseline was negative and some fluorescence was actually being produced by the assay solutions.
-		+	* The HRP concentration was further varied from trials on [[User:Melissa_Novy/Notebook/CHEM-571/2012/09/25\|2012/09/25]] in an attempt to slow the reaction rate such that the fluorimeter could detect the decline in fluorescence.
-	*~~'''~~[[User:~~Abigail E. Miller~~\|~~Abigail E. Miller~~]] ~~18:12, 7 October 2012 (EDT)''':again, included all information~~ in ~~your notebook. everything~~ in ~~the report should be pulled form your notebook~~. ~~also , where di the stock solutions come from~~?	+	* ???

		+	==Notes==
		+	* The volumes of the components added to each of the reaction solutions was not varied during the experiment. Please refer to the following table for the volumes added:
		+	{\| {{table}}
		+	\| align="center" style="background:#f0f0f0;"\|'''Solution'''
		+	\| align="center" style="background:#f0f0f0;"\|'''Volume [μL]'''
		+	\|-
		+	\| 4-Iodophenol\|\|13.2
		+	\|-
		+	\| Luminol\|\|920
		+	\|-
		+	\| H<sub>2</sub>O<sub>2</sub>\|\|990
		+	\|-
		+	\| HRP\|\|66
		+	\|}
	<!-- ##### 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. ##### -->

	__NOTOC__		__NOTOC__

Abigail E. Miller: /* Objectives */

2012-10-07T22:14:31Z

Objectives

←Older revision		Revision as of 22:14, 7 October 2012
Line 19:		Line 19:
	** Make binding buffer and elution buffer.		** Make binding buffer and elution buffer.
	** Please refer to [[User:Dhea_Patel/Notebook/Experimental_Biological_Chemistry_Notebook/2012/09/26\|Dhea Patel's entry]] for the protocols and calculations of the above solutions.		** Please refer to [[User:Dhea_Patel/Notebook/Experimental_Biological_Chemistry_Notebook/2012/09/26\|Dhea Patel's entry]] for the protocols and calculations of the above solutions.
-	*'''[[User:Abigail E. Miller\|Abigail E. Miller]] 18:09, 7 October 2012 (EDT)''':you need ot include the specifics of the ~~bidnign nad~~ elution ~~bufers~~, pH final volume and ~~concnetratiosn~~ of each component.	+	*'''[[User:Abigail E. Miller\|Abigail E. Miller]] 18:09, 7 October 2012 (EDT)''':you need to include the specifics of the binding and elution buffers, pH final volume and concentration of each component.
	* Continue optimizing the HRP chemiluminescence assay by adjusting the concentrations of the reactants.		* Continue optimizing the HRP chemiluminescence assay by adjusting the concentrations of the reactants.
	* Analyze Tris buffers at pH 8 and 10 and at various concentrations with UV-vis spectroscopy.		* Analyze Tris buffers at pH 8 and 10 and at various concentrations with UV-vis spectroscopy.
		+	*'''[[User:Abigail E. Miller\|Abigail E. Miller]] 18:14, 7 October 2012 (EDT)''':didn't you also need ot do something with the cells for making the ADA protein?

	==Spectroscopy Data of Tris Buffers==		==Spectroscopy Data of Tris Buffers==

Abigail E. Miller: /* HRP Chemiluminescence Assay */

2012-10-07T22:12:47Z

HRP Chemiluminescence Assay

←Older revision		Revision as of 22:12, 7 October 2012
Line 52:		Line 52:
	* Graph of intensity versus time of 4-iodophenol-enhanced solutions of hydrogen peroxide, luminol, and HRP.		* Graph of intensity versus time of 4-iodophenol-enhanced solutions of hydrogen peroxide, luminol, and HRP.
	* Refer to the [[Media: HRP_Assays.pdf\|report]] for analysis of HRP assays with AAP and luminol.		* Refer to the [[Media: HRP_Assays.pdf\|report]] for analysis of HRP assays with AAP and luminol.
		+
		+	*'''[[User:Abigail E. Miller\|Abigail E. Miller]] 18:12, 7 October 2012 (EDT)''':again, included all information in your notebook. everything in the report should be pulled form your notebook. also , where di the stock solutions come from?

	<!-- ##### 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. ##### -->

	__NOTOC__		__NOTOC__

Abigail E. Miller: /* Spectroscopy Data of Tris Buffers */

2012-10-07T22:11:19Z

Spectroscopy Data of Tris Buffers

←Older revision		Revision as of 22:11, 7 October 2012
Line 29:		Line 29:


-	it is not at all clear that this is tris buffer added to AuNPs. and ~~htere~~ is no link to where the AuNPs came from or when these solutions were mixed.	+	*'''[[User:Abigail E. Miller\|Abigail E. Miller]] 18:11, 7 October 2012 (EDT)''':it is not at all clear that this is tris buffer added to AuNPs. and there is no link to where the AuNPs came from or when these solutions were mixed. or is that not what this is about?

	==HRP Chemiluminescence Assay==		==HRP Chemiluminescence Assay==

Abigail E. Miller: /* Spectroscopy Data of Tris Buffers */

2012-10-07T22:10:43Z

Spectroscopy Data of Tris Buffers

←Older revision		Revision as of 22:10, 7 October 2012
Line 27:		Line 27:
	* Graph of absorbance versus wavelength of Tris buffer in H<sub>2</sub>O at pH 8 and 10 and at concentrations ranging from 10 μM to 10 mM.		* Graph of absorbance versus wavelength of Tris buffer in H<sub>2</sub>O at pH 8 and 10 and at concentrations ranging from 10 μM to 10 mM.
	* Note that very small peaks were observed around 730-740 nm for all solutions and another small, broad peak was observed around 600 nm for the 10 μM Tris buffer solution at pH 8. The data indicate that absorbance of Tris buffer does not affect the peak observed for AuNPs at around 540 nm and the concentration and pH of Tris does not significantly affect the peaks for Tris observed.		* Note that very small peaks were observed around 730-740 nm for all solutions and another small, broad peak was observed around 600 nm for the 10 μM Tris buffer solution at pH 8. The data indicate that absorbance of Tris buffer does not affect the peak observed for AuNPs at around 540 nm and the concentration and pH of Tris does not significantly affect the peaks for Tris observed.
		+
		+
		+	it is not at all clear that this is tris buffer added to AuNPs. and htere is no link to where the AuNPs came from or when these solutions were mixed.

	==HRP Chemiluminescence Assay==		==HRP Chemiluminescence Assay==

Abigail E. Miller: /* Objectives */

2012-10-07T22:09:15Z

Objectives

←Older revision		Revision as of 22:09, 7 October 2012
Line 19:		Line 19:
	** Make binding buffer and elution buffer.		** Make binding buffer and elution buffer.
	** Please refer to [[User:Dhea_Patel/Notebook/Experimental_Biological_Chemistry_Notebook/2012/09/26\|Dhea Patel's entry]] for the protocols and calculations of the above solutions.		** Please refer to [[User:Dhea_Patel/Notebook/Experimental_Biological_Chemistry_Notebook/2012/09/26\|Dhea Patel's entry]] for the protocols and calculations of the above solutions.
		+	*'''[[User:Abigail E. Miller\|Abigail E. Miller]] 18:09, 7 October 2012 (EDT)''':you need ot include the specifics of the bidnign nad elution bufers, pH final volume and concnetratiosn of each component.
	* Continue optimizing the HRP chemiluminescence assay by adjusting the concentrations of the reactants.		* Continue optimizing the HRP chemiluminescence assay by adjusting the concentrations of the reactants.
	* Analyze Tris buffers at pH 8 and 10 and at various concentrations with UV-vis spectroscopy.		* Analyze Tris buffers at pH 8 and 10 and at various concentrations with UV-vis spectroscopy.