User:Javier Vinals Camallonga/Notebook/Javier Vinals notebook/2013/10/01

From OpenWetWare

(Difference between revisions)
Jump to: navigation, search
(Autocreate 2013/10/01 Entry for User:Javier_Vinals_Camallonga/Notebook/Javier_Vinals_notebook)
(Objective)
Line 10: Line 10:
==Objective==
==Objective==
-
Today we'll be determining the molar absorptivities of two different molecules, [http://en.wikipedia.org/wiki/Adenosine adenosine] and [http://en.wikipedia.org/wiki/Inosine inosine]. The data that we generate today will be important when we study [http://en.wikipedia.org/wiki/Adenosine_deaminase adenosine deaminase] (ADA), which converts adenosine to inosine. The difference between these two molecules is that adenosine contains a primary amine whereas inosine contains a carboxy group. Overexpression of this protein causes anemia in humans. A shortage of this protein can lead to severe immuno-defficiency.  
+
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.
-
Adenosine and inosine have different absorption spectra. We will be observing changes in UV-Vis spectra to determine changes in concentration of both adenosine and inosine. In order to do this, we will need to know the molar absorptivity (ε) of both of these molecules. Just as each molecule has a characteristic absorption at each wavelength, this (per-wavelength) absorption can be quantified by a molar absorptivity. Or ... for a given concentration a molecule will absorb a very specific amount of light at a precise wavelength. A molecule doesn't have just one molar absorptivity; there is a molar absorptivity to describe each wavelength in a molecular absorbance spectrum.
+
==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

Revision as of 13:50, 1 October 2013

Project name Main project page
Previous entry      Next entry

Entry title

  • Insert content here...

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


Personal tools