User:Javier Vinals Camallonga/Notebook/Javier Vinals notebook/2013/10/01: Difference between revisions
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==Objective== | ==Objective== | ||
To monitor the kinetics and yield of the [http://en.wikipedia.org/wiki/Horseradish_peroxidase horseradish peroxidase]-catalyzed oxidation of [http://en.wikipedia.org/wiki/Luminol luminol]. These experiments will be compared to future experiments with HRP-functionalized nanoparticles. These experiments are also meant to introduce researchers to stopped-flow techniques and rapid data collection. | |||
==Description== | |||
Three sets of measurements will be performed today. | |||
# UV-Vis Absorbance of reactants, catalysts, and products | |||
## horseradish peroxidase (Use stock solution) | |||
## luminol (use stock solution) | |||
## 3-aminophthalic acid (product of reaction between luminol and H2O2 catalyzed by HRP. In order to take this measurement, react (in 1:1 ratio or with slight excess H2O2) luminol with H2O2 in presence of HRP. Allow the reaction to proceed for 5 minutes; take the spectrum) | |||
# Chemiluminescence of luminol oxidation reaction initiated by stopped flow mixer | |||
## Add HRP/Luminol stock solution to stopped flow mixer | |||
## Add H2O2 stock solution to stopped flow mixer | |||
## equilibrate mixer tubes with sample. | |||
## Initiate Mixing | |||
## Measure light produced as result of reaction, integrated over a specific time range | |||
## Integrate area under the curve | |||
# Kinetics of luminol oxidized by changes in absorption spectrum, reaction carried out in stopped flow mixer | |||
## Add HRP/Luminol stock solution to stopped flow mixer | |||
## Add H2O2 stock solution to stopped flow mixer | |||
## equilibrate mixer tubes with sample. | |||
## Initiate Mixing | |||
## Using luminol and 3-aminophthalic acid spectra as endpoints, determine the kinetics of 3-aminophthalic acid synthesis. | |||
*[[User:Matt Hartings|Matt Hartings]] Note: We will be doing Step 1 (while I'm teaching my other class) and Step 3 (after I get back from class) today. We'll do step 2 tomorrow. | |||
==Data== | |||
<u>Stock Solution</u> | |||
# Buffer | |||
## 0.6175g Tris in 1L, pH set to 8 with HCl ---> 5.1mM | |||
# HRP | |||
## 1.7mg in 50mL buffer (MW ~ 44,000) ---> 0.77uM | |||
# Luminol | |||
## Dissolve 13.4mg luminol in 300uL of DMSO | |||
## Add to 50mL buffer ---> 1.51mM | |||
# H2O2 | |||
## 177uL 30% H2O2 into 50mL buffer ---> Should be 45mM | |||
## Check this concentration! The molar absorptivity of H2O2 at 240nm is 40,000 M<sup>-1</sup>cm<sup>-1 | |||
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Entry title
ObjectiveTo monitor the kinetics and yield of the horseradish peroxidase-catalyzed oxidation of luminol. These experiments will be compared to future experiments with HRP-functionalized nanoparticles. These experiments are also meant to introduce researchers to stopped-flow techniques and rapid data collection. DescriptionThree sets of measurements will be performed today.
DataStock Solution
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