User:Javier Vinals Camallonga/Notebook/Javier Vinals notebook/2014/03/05

From OpenWetWare

(Difference between revisions)
Jump to: navigation, search
(Autocreate 2014/03/05 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.  
+
Today we'll be calculating the equilibrium constants for heated 30:1 lysozyme AuNPs with variables added after baking, and room temperature 30:1 lysozyme AuNPs. Then we will make standards with the amount of gold per 5mL tube to run AA and UV-Vis. Then we will centrifuge the samples from yesterday, and run AA and UV-Vis on centrifuged samples.
 +
 
 +
==Procedure==
 +
===Atomic Absorption===
 +
 
 +
'''Creating the Gold Stock Solutions'''
 +
# Add 50 μL of HAuCl<sub>4</sub>·3H<sub>2</sub>O and 4950 μL of distilled water to a Falcon tube, for a final concentration of 10 μg/mL Au.
 +
# Add 100 μL of HAuCl<sub>4</sub>·3H<sub>2</sub>O and 4900 μL of distilled water to a Falcon tube, for a final concentration of 20 μg/mL Au.
 +
# Add 150 μL of HAuCl<sub>4</sub>·3H<sub>2</sub>O and 4850 μL of distilled water to a Falcon tube, for a final concentration of 30 μg/mL Au.
 +
# Add 200 μL of HAuCl<sub>4</sub>·3H<sub>2</sub>O and 4800 μL of distilled water to a Falcon tube, for a final concentration of 40 μg/mL Au.
 +
# Add 250 μL of HAuCl<sub>4</sub>·3H<sub>2</sub>O and 4750 μL of distilled water to a Falcon tube, for a final concentration of 50 μg/mL Au.
 +
# A sixth stock solution of gold was created with 448 μL of HAuCl<sub>4</sub>·3H<sub>2</sub>O and 4552 μL of distilled water to a Falcon tube. This resulting concentration represents the amount of gold in a 5 mL sample of lysozyme-AuNP.
 +
 
 +
 
 +
 
 +
'''Atomic Absorption Samples'''
 +
 
 +
The following samples were run on the AA:
 +
* 30:1 lysozyme-AuNP with NaCl, KCl, MgCl<sub>2</sub>, CaCl<sub>2</sub>, 2,2 bipyridine, MES, citric acid
 +
* 60:1, 70:1, 80:1, 90:1, 100:1 BSA-AuNP with MgCl<sub>2</sub>
 +
* Gold solution in 5 mL sample of lysozyme-AuNP
 +
* Control of 30:1 lysozyme-AuNP without any added variable
-
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.
 

Revision as of 19:21, 16 March 2014

Project name Main project page
Previous entry      Next entry

Entry title

  • Insert content here...

Objective

Today we'll be calculating the equilibrium constants for heated 30:1 lysozyme AuNPs with variables added after baking, and room temperature 30:1 lysozyme AuNPs. Then we will make standards with the amount of gold per 5mL tube to run AA and UV-Vis. Then we will centrifuge the samples from yesterday, and run AA and UV-Vis on centrifuged samples.

Procedure

Atomic Absorption

Creating the Gold Stock Solutions

  1. Add 50 μL of HAuCl4·3H2O and 4950 μL of distilled water to a Falcon tube, for a final concentration of 10 μg/mL Au.
  2. Add 100 μL of HAuCl4·3H2O and 4900 μL of distilled water to a Falcon tube, for a final concentration of 20 μg/mL Au.
  3. Add 150 μL of HAuCl4·3H2O and 4850 μL of distilled water to a Falcon tube, for a final concentration of 30 μg/mL Au.
  4. Add 200 μL of HAuCl4·3H2O and 4800 μL of distilled water to a Falcon tube, for a final concentration of 40 μg/mL Au.
  5. Add 250 μL of HAuCl4·3H2O and 4750 μL of distilled water to a Falcon tube, for a final concentration of 50 μg/mL Au.
  6. A sixth stock solution of gold was created with 448 μL of HAuCl4·3H2O and 4552 μL of distilled water to a Falcon tube. This resulting concentration represents the amount of gold in a 5 mL sample of lysozyme-AuNP.


Atomic Absorption Samples

The following samples were run on the AA:

  • 30:1 lysozyme-AuNP with NaCl, KCl, MgCl2, CaCl2, 2,2 bipyridine, MES, citric acid
  • 60:1, 70:1, 80:1, 90:1, 100:1 BSA-AuNP with MgCl2
  • Gold solution in 5 mL sample of lysozyme-AuNP
  • Control of 30:1 lysozyme-AuNP without any added variable



Personal tools