User:Melissa Novy/Notebook/CHEM-571/2012/11/07 - Revision history

Melissa Novy: /* Bradford Assay */

Melissa Novy — Mon, 26 Nov 2012 20:13:14 GMT

Bradford Assay

←Older revision		Revision as of 20:13, 26 November 2012
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	<br>		<br>
	* Seven standard solutions were prepared with 1X Bradford reagent, BSA stock solution, and autoclaved water, and then analyzed with UV-vis from 500 to 800 nm.		* Seven standard solutions were prepared with 1X Bradford reagent, BSA stock solution, and autoclaved water, and then analyzed with UV-vis from 500 to 800 nm.
		+	** Note that Bradford reagent changes color from red to blue in the presence of protein, as it binds to the protein backbone. The absorbance of Bradford reagent depends on the concentration of protein in solution.
	* Note that the original solutions as listed below produced absorbances over 1, such that the resultant calibration curve would likely not be linear. All solutions were diluted by half, by adding 715 μL of the original solution to 715 μL of autoclaved water.		* Note that the original solutions as listed below produced absorbances over 1, such that the resultant calibration curve would likely not be linear. All solutions were diluted by half, by adding 715 μL of the original solution to 715 μL of autoclaved water.
	{\| {{table}}		{\| {{table}}

Melissa Novy: /* Objectives */

Melissa Novy — Mon, 26 Nov 2012 20:07:19 GMT

Objectives

←Older revision		Revision as of 20:07, 26 November 2012
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	==Objectives==		==Objectives==
-	* Conduct a Bradford assay on fractions of ADA, made on [[User:Melissa_Novy/Notebook/CHEM-571/2012/10/03\|2012/10/03]] and [[User:Melissa_Novy/Notebook/CHEM-571/2012/11/06\|2012/11/06]].	+	* Conduct a Bradford assay on fractions of ADA, made on [[User:Melissa_Novy/Notebook/CHEM-571/2012/10/03\|2012/10/03]] and [[User:Melissa_Novy/Notebook/CHEM-571/2012/11/06\|2012/11/06]] to determine the concentration of ADA in the fractions.

	==Bradford Assay==		==Bradford Assay==

Melissa Novy: /* Objectives */

Melissa Novy — Thu, 08 Nov 2012 18:13:07 GMT

Objectives

←Older revision		Revision as of 18:13, 8 November 2012
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	==Objectives==		==Objectives==
-	* Conduct Bradford assay on fractions of ADA, made on [[User:Melissa_Novy/Notebook/CHEM-571/2012/10/03\|2012/10/03]] and [[User:Melissa_Novy/Notebook/CHEM-571/2012/11/06\|2012/11/06]].	+	* Conduct a Bradford assay on fractions of ADA, made on [[User:Melissa_Novy/Notebook/CHEM-571/2012/10/03\|2012/10/03]] and [[User:Melissa_Novy/Notebook/CHEM-571/2012/11/06\|2012/11/06]].

-		+	==Bradford Assay==
-	<!-- ~~##### DO NOT edit~~ below ~~this line unless you know what you are doing~~. ##### -->	+	* A stock solution of 1 mg/mL of BSA was prepared by adding 10 mg of BSA into a 10 mL volumetric flask, then filling it with autoclaved water.
		+	** 102 mg of BSA was added into a 10 mL volumetric flask, giving an actual concentration of 1.02 mg/mL BSA in autoclaved water.
		+	<br>
		+	* Seven standard solutions were prepared with 1X Bradford reagent, BSA stock solution, and autoclaved water, and then analyzed with UV-vis from 500 to 800 nm.
		+	* Note that the original solutions as listed below produced absorbances over 1, such that the resultant calibration curve would likely not be linear. All solutions were diluted by half, by adding 715 μL of the original solution to 715 μL of autoclaved water.
		+	{\| {{table}}
		+	\| align="center" style="background:#f0f0f0;"\|'''Volume 1X Bradford Reagent Added [mL]'''
		+	\| align="center" style="background:#f0f0f0;"\|'''Volume BSA Stock Added [mL]'''
		+	\| align="center" style="background:#f0f0f0;"\|'''Volume Water Added [mL]'''
		+	\| align="center" style="background:#f0f0f0;"\|'''Conc. BSA [mg/mL]'''
		+	\|-
		+	\| 1.4\|\|0\|\|0.03\|\|0
		+	\|-
		+	\| 1.4\|\|0.005\|\|0.025\|\|0.003566434
		+	\|-
		+	\| 1.4\|\|0.01\|\|0.02\|\|0.007132867
		+	\|-
		+	\| 1.4\|\|0.015\|\|0.015\|\|0.010699301
		+	\|-
		+	\| 1.4\|\|0.02\|\|0.01\|\|0.014265734
		+	\|-
		+	\| 1.4\|\|0.025\|\|0.005\|\|0.017832168
		+	\|-
		+	\| 1.4\|\|0.03\|\|0\|\|0.021398601
	\|}		\|}
		+	<br>
		+	* The absorbance values at 595 nm of each solution were graphed versus BSA concentration in mg/mL to produce a calibration curve.
		+	* Using linear regression, the equation of the line was obtained.
		+	[[Image:Bradford_assay.png]]
		+	<br>
		+	* Fractions of ADA, made on [[User:Melissa_Novy/Notebook/CHEM-571/2012/10/03\|2012/10/03]] and [[User:Melissa_Novy/Notebook/CHEM-571/2012/11/06\|2012/11/06]], were obtained.
		+	* 7.5 μL of each fraction was added to 7.5 μL of autoclaved water and 715 μL of 1X Bradford reagent.
		+	** Fraction #2, obtained in November, was diluted by half, using the same procedure as listed above for the standard solutions, such that its absorbance was below 1.
		+	{\| {{table}}
		+	\| align="center" style="background:#f0f0f0;"\|'''Sample Name'''
		+	\| align="center" style="background:#f0f0f0;"\|'''Volume of ADA [mL]'''
		+	\| align="center" style="background:#f0f0f0;"\|'''Absorbance []'''
		+	\| align="center" style="background:#f0f0f0;"\|'''Conc. ADA [mg/mL]'''
		+	\| align="center" style="background:#f0f0f0;"\|'''Mass of ADA [mg]'''
		+	\| align="center" style="background:#f0f0f0;"\|'''Moles of ADA [mol]'''
		+	\| align="center" style="background:#f0f0f0;"\|'''Molar Absorptivity of ADA [mL/mg cm]'''
		+	\| align="center" style="background:#f0f0f0;"\|'''Molarity [M]'''
		+	\|-
		+	\| Fraction Oct\|\|0.0075\|\|0.784\|\|0.012459254\|\|0.017816733\|\|4.37758E-10\|\|62.92511628\|\|5.83677E-05
		+	\|-
		+	\| Fraction 2\|\|0.00325\|\|0.559\|\|0.004310033\|\|0.006163347\|\|1.51434E-10\|\|129.697395\|\|4.65949E-05
		+	\|-
		+	\| Fraction 1&3\|\|0.0075\|\|0.501\|\|0.002209344\|\|0.003159363\|\|7.76256E-11\|\|226.7640984\|\|1.03501E-05
		+	\|}
		+	<!-- ##### DO NOT edit below this line unless you know what you are doing. ##### -->

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Melissa Novy at 17:54, 8 November 2012

Melissa Novy — Thu, 08 Nov 2012 17:54:46 GMT

←Older revision		Revision as of 17:54, 8 November 2012
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	<!-- ##### DO NOT edit above this line unless you know what you are doing. ##### -->		<!-- ##### DO NOT edit above this line unless you know what you are doing. ##### -->

-	==~~Entry title~~==	+	==Objectives==
-	* ~~Insert content here..~~.	+	* Conduct Bradford assay on fractions of ADA, made on [[User:Melissa_Novy/Notebook/CHEM-571/2012/10/03\|2012/10/03]] and [[User:Melissa_Novy/Notebook/CHEM-571/2012/11/06\|2012/11/06]].

Melissa Novy: Autocreate 2012/11/07 Entry for User:Melissa_Novy/Notebook/CHEM-571

Melissa Novy — Thu, 08 Nov 2012 17:52:00 GMT

Autocreate 2012/11/07 Entry for User:Melissa_Novy/Notebook/CHEM-571

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