User:Keyun Wang/Notebook/Experimental Biological Chemistry I/2012/11/07 - Revision history

Keyun Wang: /* Procedure for Making Au/HRP samples */

Keyun Wang — Sat, 08 Dec 2012 04:05:51 GMT

Procedure for Making Au/HRP samples

←Older revision		Revision as of 04:05, 8 December 2012
Line 97:		Line 97:
	\| align="center" style="background:#f0f0f0;"\|'''HAuCl4 added [uL]'''		\| align="center" style="background:#f0f0f0;"\|'''HAuCl4 added [uL]'''
	\| align="center" style="background:#f0f0f0;"\|'''Water added [uL]'''		\| align="center" style="background:#f0f0f0;"\|'''Water added [uL]'''
		+	\| align="center" style="background:#f0f0f0;"\|'''Final concentration of Au[uM]'''
		+	\| align="center" style="background:#f0f0f0;"\|'''Final concentration of HRP[uM]'''
	\|-		\|-
-	\| 10\|\|280.7\|\|7.62\|\|7711.68	+	\| 10\|\|280.7\|\|7.62\|\|7711.68\|\|1\|\|10
	\|-		\|-
-	\| 50\|\|280.7\|\|38.1\|\|7681.2	+	\| 50\|\|280.7\|\|38.1\|\|7681.2\|\|1\|\|50
	\|-		\|-
-	\| 100\|\|280.7\|\|76.2\|\|7643.1	+	\| 100\|\|280.7\|\|76.2\|\|7643.1\|\|1\|\|100
	\|-		\|-
-	\| 150\|\|280.7\|\|114.3\|\|7605	+	\| 150\|\|280.7\|\|114.3\|\|7605\|\|1\|\|150
	\|-		\|-
-	\| 200\|\|280.7\|\|152.4\|\|7566.9	+	\| 200\|\|280.7\|\|152.4\|\|7566.9\|\|1\|\|200
	\|-		\|-
-	\| 250\|\|280.7\|\|190.5\|\|7528.8	+	\| 250\|\|280.7\|\|190.5\|\|7528.8\|\|1\|\|250
	\|-		\|-
-	\| 300\|\|280.7\|\|228.5\|\|7490.8	+	\| 300\|\|280.7\|\|228.5\|\|7490.8\|\|1\|\|300
	\|-		\|-
-	\| 350\|\|280.7\|\|266.6\|\|7452.7	+	\| 350\|\|280.7\|\|266.6\|\|7452.7\|\|1\|\|350
	\|-		\|-
-	\| 400\|\|280.7\|\|304.7\|\|7414.6	+	\| 400\|\|280.7\|\|304.7\|\|7414.6\|\|1\|\|400
	\|-		\|-
-	\| 450\|\|280.7\|\|342.8\|\|7376.5	+	\| 450\|\|280.7\|\|342.8\|\|7376.5\|\|1\|\|450
	\|}		\|}
	* Each Au/HRP samples were capped, wrapped with aluminum foil, and placed in incubator at 85°C for 4 hours then cooled down to room temperature.		* Each Au/HRP samples were capped, wrapped with aluminum foil, and placed in incubator at 85°C for 4 hours then cooled down to room temperature.

Keyun Wang: /* Procedure for Making Au/HRP samples */

Keyun Wang — Wed, 28 Nov 2012 05:38:45 GMT

Procedure for Making Au/HRP samples

←Older revision		Revision as of 05:38, 28 November 2012
Line 87:		Line 87:
	* Au/HRP samples were made with the mole ratios listed below:		* Au/HRP samples were made with the mole ratios listed below:
	10 - 50 - 100 - 150 - 200 - 250 - 300 - 350 - 400 - 450		10 - 50 - 100 - 150 - 200 - 250 - 300 - 350 - 400 - 450
-	*	+	* 28.5uM HRP stock solution was made. HRP from Alfa Aesar® has a molecular weight of 40,000 g/mol. 0.0057g of HRP was weighted out and mixed with 5mL of distilled water. The following calculations was done to obtain masses weighted for 28.5uM HRP stock solution:
		+	28.5uM HRP * 0.005L = 1.5E-7 mol
		+	1.5E-7mol * 40,000g/mol = 0.0057g of HRP
		+	* HAuCl<sub>4</sub> used for making Au/HRP samples were made on [[User:Keyun Wang/Notebook/Experimental Biological Chemistry I/2012/09/05\|09/05/2012]] and has a concentration of 10.5mM.
		+	* Volume of HRP was set at 280.7uL while volumes of HAuCl<sub>4</sub> were ranging from 7.62μL to 342.8μL. Distilled water was added until volume of sample at each mole ratio reached 8mL. Detailed volumes for each reactant is listed in table below:
		+	{\| {{table}}
		+	\| align="center" style="background:#f0f0f0;"\|'''Au/HRP ratio'''
		+	\| align="center" style="background:#f0f0f0;"\|'''HRP added [uL]'''
		+	\| align="center" style="background:#f0f0f0;"\|'''HAuCl4 added [uL]'''
		+	\| align="center" style="background:#f0f0f0;"\|'''Water added [uL]'''
		+	\|-
		+	\| 10\|\|280.7\|\|7.62\|\|7711.68
		+	\|-
		+	\| 50\|\|280.7\|\|38.1\|\|7681.2
		+	\|-
		+	\| 100\|\|280.7\|\|76.2\|\|7643.1
		+	\|-
		+	\| 150\|\|280.7\|\|114.3\|\|7605
		+	\|-
		+	\| 200\|\|280.7\|\|152.4\|\|7566.9
		+	\|-
		+	\| 250\|\|280.7\|\|190.5\|\|7528.8
		+	\|-
		+	\| 300\|\|280.7\|\|228.5\|\|7490.8
		+	\|-
		+	\| 350\|\|280.7\|\|266.6\|\|7452.7
		+	\|-
		+	\| 400\|\|280.7\|\|304.7\|\|7414.6
		+	\|-
		+	\| 450\|\|280.7\|\|342.8\|\|7376.5
		+	\|}
		+	* Each Au/HRP samples were capped, wrapped with aluminum foil, and placed in incubator at 85°C for 4 hours then cooled down to room temperature.

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

Keyun Wang at 05:19, 28 November 2012

Keyun Wang — Wed, 28 Nov 2012 05:19:59 GMT

←Older revision		Revision as of 05:19, 28 November 2012
Line 10:		Line 10:
	* Au/HRP samples were made at the following mole ratios: 10, 50, 100, 150, 200, 250, 300, 350, 400, 450.		* Au/HRP samples were made at the following mole ratios: 10, 50, 100, 150, 200, 250, 300, 350, 400, 450.

-	==Procedure==	+	==Procedure for Bradford Assay==
		+	* BSA from Signma Aldrich® was used to make a stock solution of 1mg/mL. BSA stock solution was made the following way:
		+	** 10mg of BSA was weighted out
		+	** 10mg of BSA was poured into a 10mL volumetric flask
		+	** 10mL of sterile water was added to the 10ml volumetric flask
		+	** BSA and water were mixed until dissolved
		+	* 6X Bradford reagent from Bio Rad® was diluted 6 fold to make 1X Bradford reagent. 1X Bradford was diluted by mixing 4mL of 6X Bradford reagent with 20mL of sterile water. 1X Bradford reagent was used to make Bradford assay standard solutions.
		+	* Standard solutions were made with different amounts of 1mg/mL BSA, 1X bradford reagent, and water. All sample solutions made up a total volume of 1.7mL to place in plastic cuvette. The volume of each reagent used are listed in table below:
		+	{\| {{table}}
		+	\| align="center" style="background:#f0f0f0;"\|'''BSA added[mL]'''
		+	\| align="center" style="background:#f0f0f0;"\|'''1X Bradford Reagent added[mL]'''
		+	\| align="center" style="background:#f0f0f0;"\|'''Sterile Water added[mL]'''
		+	\|-
		+	\| 0\|\|1.4\|\|0.03
		+	\|-
		+	\| 0.005\|\|1.4\|\|0.025
		+	\|-
		+	\| 0.01\|\|1.4\|\|0.02
		+	\|-
		+	\| 0.015\|\|1.4\|\|0.015
		+	\|-
		+	\| 0.02\|\|1.4\|\|0.01
		+	\|-
		+	\| 0.025\|\|1.4\|\|0.005
		+	\|-
		+	\| 0.03\|\|1.4\|\|0
		+	\|}
		+	* Each samples in plastic cuvette were placed in UV-vis spectrometer under spectrum method with wavelength ranging from 500nm to 800nm. The absorbance was recorded for analysis.
		+	* After the Bradford assay were run, 7.5μL of ADA protein fractions were mixed with 715μL 1X Bradford assay and 7.5μL water to test absorbance at 595nm.

		+	==Results and Calculations of ADA protein Concentrations==
		+	* The calculated final BSA concentration is shown in table below:
		+	{\| {{table}}
		+	\| align="center" style="background:#f0f0f0;"\|'''Volume BSA Stock Added [mL]'''
		+	\| align="center" style="background:#f0f0f0;"\|'''VolumeTotal [mL]'''
		+	\| align="center" style="background:#f0f0f0;"\|'''[BSA] [mg/mL]'''
		+	\|-
		+	\| 0\|\|1.7\|\|0
		+	\|-
		+	\| 0.005\|\|1.7\|\|0.003566434
		+	\|-
		+	\| 0.01\|\|1.7\|\|0.007132867
		+	\|-
		+	\| 0.015\|\|1.7\|\|0.010699301
		+	\|-
		+	\| 0.02\|\|1.7\|\|0.014265734
		+	\|-
		+	\| 0.025\|\|1.7\|\|0.017832168
		+	\|-
		+	\| 0.03\|\|1.7\|\|0.021398601
		+	\|}
		+	* Absorbance at 595nm were plotted on a graph against the final concentration of BSA used before putting into UV-vis spectrometer. The graph is shown below:
		+	[[Image:Bradford_assay.png\|600px]]
		+	* Best fit line was shown in graph as function to calculate ADA protein concentration once absorbance was measured. The absorbance for ADA protein was kept below 1 to make sure measurement was within the limit of Bradford assay.
		+	* With the calculated best fit line, the absorbance of each protein fraction was used to calculate ADA protein concentration by fitting into the equation:
		+	Absorbance = 27.609 * [BSA]mg/mL + 0.4363
		+	* Table including the absorbance obtained and final calculated ADA protein concentration in molarity is listed below:
		+	{\| {{table}}
		+	\| align="center" style="background:#f0f0f0;"\|'''-'''
		+	\| 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\|\|0.784\|\|0.012459254\|\|0.017816733\|\|4.38E-10\|\|62.92511628\|\|5.84E-05
		+	\|-
		+	\| Fraction 2\|\|0.559\|\|0.004310033\|\|0.006163347\|\|1.51E-10\|\|129.697395\|\|4.66E-05
		+	\|-
		+	\| Fraction 1&3\|\|0.501\|\|0.002209344\|\|0.003159363\|\|7.76E-11\|\|226.7640984\|\|1.04E-05
		+	\|}
		+	* Thus, the final concentration of ADA protein fraction made on [[User:Keyun Wang/Notebook/Experimental Biological Chemistry I/2012/10/03\|10/03/2012]] is 58.4mM. Final concentration of ADA protein fraction 2 made on [[User:Keyun Wang/Notebook/Experimental Biological Chemistry I/2012/11/06\|11/06/2012]] is 46.6mM. And final concentration of ADA protein fraction 1&3 made on the same day is 10.4mM.

		+	==Procedure for Making Au/HRP samples==
		+	* Au/HRP samples were made with the mole ratios listed below:
		+	10 - 50 - 100 - 150 - 200 - 250 - 300 - 350 - 400 - 450
		+	*

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

Keyun Wang at 04:39, 28 November 2012

Keyun Wang — Wed, 28 Nov 2012 04:39:37 GMT

←Older revision		Revision as of 04:39, 28 November 2012
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	{\|{{table}} width="800"		{\|{{table}} width="800"
	\|-		\|-
-	\|style="background-color: #EEE"\|[[Image:owwnotebook_icon.png\|128px]]<span style="font-size:22px;"> ~~Project name~~</span>	+	\|style="background-color: #EEE"\|[[Image:owwnotebook_icon.png\|128px]]<span style="font-size:22px;"> Experimental Biological Chemistry I</span>
	\|style="background-color: #F2F2F2" align="center"\|<html><img src="/images/9/94/Report.png" border="0" /></html> [[{{#sub:{{FULLPAGENAME}}\|0\|-11}}\|Main project page]]<br />{{#if:{{#lnpreventry:{{FULLPAGENAME}}}}\|<html><img src="/images/c/c3/Resultset_previous.png" border="0" /></html>[[{{#lnpreventry:{{FULLPAGENAME}}}}{{!}}Previous entry]]<html>      </html>}}{{#if:{{#lnnextentry:{{FULLPAGENAME}}}}\|[[{{#lnnextentry:{{FULLPAGENAME}}}}{{!}}Next entry]]<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html>}}		\|style="background-color: #F2F2F2" align="center"\|<html><img src="/images/9/94/Report.png" border="0" /></html> [[{{#sub:{{FULLPAGENAME}}\|0\|-11}}\|Main project page]]<br />{{#if:{{#lnpreventry:{{FULLPAGENAME}}}}\|<html><img src="/images/c/c3/Resultset_previous.png" border="0" /></html>[[{{#lnpreventry:{{FULLPAGENAME}}}}{{!}}Previous entry]]<html>      </html>}}{{#if:{{#lnnextentry:{{FULLPAGENAME}}}}\|[[{{#lnnextentry:{{FULLPAGENAME}}}}{{!}}Next entry]]<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html>}}
	\|-		\|-
	\| colspan="2"\|		\| colspan="2"\|
	<!-- ##### 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~~==	+	==Purpose==
-	* ~~Insert content here..~~.	+	* Bradford assay was run to test the concentration of ADA protein purified through FPLC on [[User:Keyun Wang/Notebook/Experimental Biological Chemistry I/2012/10/03\|10/03/2012]] and [[User:Keyun Wang/Notebook/Experimental Biological Chemistry I/2012/11/06\|11/06/2012]]
		+	* Au/HRP samples were made at the following mole ratios: 10, 50, 100, 150, 200, 250, 300, 350, 400, 450.
		+
		+	==Procedure==
		+

Keyun Wang: Autocreate 2012/11/07 Entry for User:Keyun_Wang/Notebook/Experimental_Biological_Chemistry_I

Keyun Wang — Wed, 28 Nov 2012 04:06:31 GMT

Autocreate 2012/11/07 Entry for User:Keyun_Wang/Notebook/Experimental_Biological_Chemistry_I

New page

{|{{table}} width="800"
|-
|style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;"> Project name</span>
|style="background-color: #F2F2F2" align="center"|<html><img src="/images/9/94/Report.png" border="0" /></html> [[{{#sub:{{FULLPAGENAME}}|0|-11}}|Main project page]]<br />{{#if:{{#lnpreventry:{{FULLPAGENAME}}}}|<html><img src="/images/c/c3/Resultset_previous.png" border="0" /></html>[[{{#lnpreventry:{{FULLPAGENAME}}}}{{!}}Previous entry]]<html>      </html>}}{{#if:{{#lnnextentry:{{FULLPAGENAME}}}}|[[{{#lnnextentry:{{FULLPAGENAME}}}}{{!}}Next entry]]<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html>}}
|-
| colspan="2"|

==Entry title==
* Insert content here...


|}

__NOTOC__