User:Keyun Wang/Notebook/Experimental Biological Chemistry I/2012/11/14: Difference between revisions
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** The HAuCl<sub>4</sub> stock solutions was made on [[User:Keyun Wang/Notebook/Experimental Biological Chemistry I/2012/09/05|09/05/2012]] and has a concentration of 10.50mM. | ** The HAuCl<sub>4</sub> stock solutions was made on [[User:Keyun Wang/Notebook/Experimental Biological Chemistry I/2012/09/05|09/05/2012]] and has a concentration of 10.50mM. | ||
** The water used was distilled water at 16 mega ohms. | ** The water used was distilled water at 16 mega ohms. | ||
** ADA and HAuCl<sub>4</sub> were added first, then water was added. All samples were wrapped with aluminum foil around to minimize exposure to light, and placed in 85°C incubator for 4 hours. After 4 hours of incubation, the samples were left in room temperature. | |||
** Below table listed the amount of ADA, HAuCl<sub>4</sub>, and water added for samples for all ratios: | ** Below table listed the amount of ADA, HAuCl<sub>4</sub>, and water added for samples for all ratios: | ||
{| {{table}} | |||
| align="center" style="background:#f0f0f0;"|'''Au/ADA ratio''' | |||
| align="center" style="background:#f0f0f0;"|'''ADA added[uL]''' | |||
| align="center" style="background:#f0f0f0;"|'''HAuCl<sub>4</sub>[uL]''' | |||
| align="center" style="background:#f0f0f0;"|'''Water[uL]''' | |||
|- | |||
| 60||137.1||45.71||7817.2 | |||
|- | |||
| 70||137.1||53.3||7809.6 | |||
|- | |||
| 80||137.1||60.9||7802.0 | |||
|- | |||
| 90||137.1||68.6||7794.4 | |||
|- | |||
| 100||137.1||76.2||7786.8 | |||
|- | |||
| 110||137.1||83.8||7779.1 | |||
|- | |||
| 120||137.1||91.4||7771.5 | |||
|- | |||
| 130||137.1||99.0||7763.9 | |||
|- | |||
| 140||137.1||106.7||7756.3 | |||
|- | |||
| 150||137.1||114.3||7748.7 | |||
|} | |||
* The Au/HRP solutions were also made in the same ratio as Au/ADA, ranging from 60 to 120 in increments of 10. | |||
** Sample with ratio 130, 140 and 150 were made with newly made HRP stock solution, details of making the HRP stock solution and determination of the concentration were described later on. | |||
** The HAuCl<sub>4</sub> stock solution was prepared from [[User:Keyun Wang/Notebook/Experimental Biological Chemistry I/2012/09/05|09/05/2012]] with a concentration of 10.50mM. | |||
** The HRP stock solution was made on [[User:Keyun Wang/Notebook/Experimental Biological Chemistry I/2012/09/05|09/05/2012]] and by mixing 0.0057mg of HRP with 5mL of autoclaved water. | |||
** Water used to make Au/HRP solution were deionized through 16 mega ohms filter. | |||
** HAuCl<sub>4</sub> and HRP were placed in small test tubes first, then water were added. The samples were wrapped with aluminum foil and placed in incubator under the same protocol as Au/ADA samples. | |||
** The volumes of HAuCl<sub>4</sub>, HRP, and water were 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[uL]''' | |||
| align="center" style="background:#f0f0f0;"|'''Water[uL]''' | |||
|- | |||
| 60||280.7||45.71||7673.6 | |||
|- | |||
| 70||280.7||53.3||7666.0 | |||
|- | |||
| 80||280.7||60.9||7658.4 | |||
|- | |||
| 90||280.7||68.6||7650.7 | |||
|- | |||
| 100||280.7||76.2||7643.1 | |||
|- | |||
| 110||280.7||83.8||7635.5 | |||
|- | |||
| 120||280.7||91.4||7627.9 | |||
|} | |||
* For Au/HRP samples with the following ratios were made with newly made HRP stock solution: | |||
130 - 140 - 150 | |||
* HRP stock solution were made by mixing 0.0057g of HRP with 5mL of distilled water. | |||
** However, after mixing, it was suspected that the balance for weighting out the HRP were inaccurate. As a result, UV-vis spectrometer was used. A spectrum from 600nm to 200nm were run with 200uL of newly made HRP sample running against 200uL of water in 10.00mm quartz cuvette. | |||
** At a wavelength of 402nm, the absorbance of HRP sample was 2.100. | |||
** With given molar absorptivity at 402nm (102 M*cm<sup>-1</sup>), the concentration of HRP were calculated in the following way: | |||
Absorbance = Molar absorptivity * Path length * Concentration | |||
2.100 = 102 M*cm<sup>-1</sup> * 1cm * Concentration | |||
Concentration of HRP sample = 20.58uM | |||
* With the newly calculated concentration, the volume for HAuCl<sub>4</sub> and HRP in sample Au/HRP ratio 130, 140, and 150 were recalculated, and volume of for each samples were shown below: | |||
Revision as of 19:34, 14 November 2012
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Purpose
Procedure
60 - 70 - 80 - 90 - 100 - 110 - 120 - 130 - 140 - 150
130 - 140 - 150
Absorbance = Molar absorptivity * Path length * Concentration 2.100 = 102 M*cm-1 * 1cm * Concentration Concentration of HRP sample = 20.58uM
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