User:Andrew K Farag/Notebook/Andrewfarag

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|style="background-color: #EEE"|[[Image:BDLlogo_notext_lr.png|128px]]<span style="font-size:22px;"> Biomaterials Design Lab</span>
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|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>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</html>}}{{#if:{{#lnnextentry:{{FULLPAGENAME}}}}|[[{{#lnnextentry:{{FULLPAGENAME}}}}{{!}}Next entry]]<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html>}}
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==Project Description/Abstract==
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* Place short description of project or notes regarding this project
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* Lorem ipsum dolor sit amet, consectetuer adipiscing elit. Donec porta commodo tellus. Nam a est eget libero mollis tincidunt. Aliquam purus. Quisque nulla ligula, facilisis in, pulvinar sed, molestie a, quam. Vestibulum at pede. In in sem eget odio eleifend placerat. Phasellus ultricies felis quis sapien. Etiam molestie volutpat quam. Praesent pulvinar scelerisque mi. Nam mi urna, fringilla eu, mattis sed, venenatis id, nunc. Maecenas velit eros, congue ut, placerat in, ornare vel, sem. Aenean porta enim sit amet felis gravida posuere. Phasellus faucibus nibh et orci.
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==Objective==
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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.
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==Description==
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Three sets of measurements will be performed today.
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# UV-Vis Absorbance of reactants, catalysts, and products
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## horseradish peroxidase (Use stock solution)
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## luminol (use stock solution)
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## 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)
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# Chemiluminescence of luminol oxidation reaction initiated by stopped flow mixer
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## Add HRP/Luminol stock solution to stopped flow mixer
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## Add H2O2 stock solution to stopped flow mixer
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## equilibrate mixer tubes with sample.  
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## Initiate Mixing
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## Measure light produced as result of reaction, integrated over a specific time range
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## Integrate area under the curve
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# Kinetics of luminol oxidized by changes in absorption spectrum, reaction carried out in stopped flow mixer
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## Add HRP/Luminol stock solution to stopped flow mixer
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## Add H2O2 stock solution to stopped flow mixer
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## equilibrate mixer tubes with sample.
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## Initiate Mixing
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## Using luminol and 3-aminophthalic acid spectra as endpoints, determine the kinetics of 3-aminophthalic acid synthesis.
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*[[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.
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==Data==
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<u>Stock Solution</u>
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# Buffer
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## 0.6175g Tris in 1L, pH set to 8 with HCl ---> 5.1mM
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# HRP
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## 1.7mg in 50mL buffer (MW ~ 44,000) ---> 0.77uM
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# Luminol
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## Dissolve 13.4mg luminol in 300uL of DMSO
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## Add to 50mL buffer ---> 1.51mM
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# H2O2
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## 177uL 30% H2O2 into 50mL buffer ---> Should be 45mM
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## Check this concentration! The molar absorptivity of H2O2 at 240nm is 40,000 M<sup>-1</sup>cm<sup>-1</sup>
==Notes==
==Notes==
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* Place some notes here for visitors
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Luminol is diluted to 8.3%. Original concentration of luminol is 1.5 mM. Final concentration of luminol is M=(1.5mM)(90uL)/(1080uL)=0.126mM. To get a stock solution of 10 mL of diluted luminol, 834 uL of concentrated luminol is used.
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* Example: This project is currently on hold until further notice.
 
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Use categories like tags. Change the "Course" category to the one corresponding to your course. The "Miscellaneous" tag can be used for particular experiments, as instructed by your professor. Please be sure to change or delete this tag as required so that the categories remain well organized.
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[[Category:Miscellaneous]]
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[[Category:Miscellaneous]]
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__NOTOC__
__NOTOC__

Current revision

Biomaterials Design Lab Main project page



Objective

To 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.

Description

Three sets of measurements will be performed today.

  1. UV-Vis Absorbance of reactants, catalysts, and products
    1. horseradish peroxidase (Use stock solution)
    2. luminol (use stock solution)
    3. 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)
  2. Chemiluminescence of luminol oxidation reaction initiated by stopped flow mixer
    1. Add HRP/Luminol stock solution to stopped flow mixer
    2. Add H2O2 stock solution to stopped flow mixer
    3. equilibrate mixer tubes with sample.
    4. Initiate Mixing
    5. Measure light produced as result of reaction, integrated over a specific time range
    6. Integrate area under the curve
  3. Kinetics of luminol oxidized by changes in absorption spectrum, reaction carried out in stopped flow mixer
    1. Add HRP/Luminol stock solution to stopped flow mixer
    2. Add H2O2 stock solution to stopped flow mixer
    3. equilibrate mixer tubes with sample.
    4. Initiate Mixing
    5. Using luminol and 3-aminophthalic acid spectra as endpoints, determine the kinetics of 3-aminophthalic acid synthesis.
  • 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

Stock Solution

  1. Buffer
    1. 0.6175g Tris in 1L, pH set to 8 with HCl ---> 5.1mM
  2. HRP
    1. 1.7mg in 50mL buffer (MW ~ 44,000) ---> 0.77uM
  3. Luminol
    1. Dissolve 13.4mg luminol in 300uL of DMSO
    2. Add to 50mL buffer ---> 1.51mM
  4. H2O2
    1. 177uL 30% H2O2 into 50mL buffer ---> Should be 45mM
    2. Check this concentration! The molar absorptivity of H2O2 at 240nm is 40,000 M-1cm-1

Notes

Luminol is diluted to 8.3%. Original concentration of luminol is 1.5 mM. Final concentration of luminol is M=(1.5mM)(90uL)/(1080uL)=0.126mM. To get a stock solution of 10 mL of diluted luminol, 834 uL of concentrated luminol is used.


Use categories like tags. Change the "Course" category to the one corresponding to your course. The "Miscellaneous" tag can be used for particular experiments, as instructed by your professor. Please be sure to change or delete this tag as required so that the categories remain well organized.




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