User:Javier Vinals Camallonga/Notebook/Javier Vinals notebook/2013/09/18: Difference between revisions

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==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.
We're going to perform a redox titration on HRP in order to determine the standard potential of this protein.
 
==Description==
We will use a [http://www.pineinst.com/echem/viewproduct.asp?ID=47955 Pine Instruments Honeycomb Spectroelectrochemical cell] coupled to a [http://www.pineinst.com/echem/viewproduct.asp?ID=47071 WaveNow USB Potentiostat]. The redox reaction will be monitored using a [http://en.wikipedia.org/wiki/Galvanic_cell galvanic cell] setting. UV Vis spectra will be recorded on an [http://www.oceanoptics.com/Products/jaz.asp Ocean Optics Jaz Spectrometer]. We will specifically use the Q-band to observe redox state following with my results from [[User:Matt_Hartings/Notebook/AU_Biomaterials_Design_Lab/2013/09/17|yesterday]].
 
Following the procedure in [http://pubs.acs.org/doi/abs/10.1021/bi9816857 this reference], we will add DTT in 1uL increments and observe both the UVVis spectrum and the open circuit potential of the SpecEchem cell. We will ultimately plot %oxidized or %reduced versus voltage read.
 
The degassed buffer will contain:
# 50mM Tris
# 50mM NaCl
and the following redox mediators (in order to stabilize the solution potential)
# 20uM duroquinone (tetramethyl-1,4-benzoquinone) (for 20mM should measure 32.8mg in 10mL)
# <strike>10uM pyocyanine</strike> (we didn't receive this chemical)
# 10uM 2-hydroxy-1,4-naphthoquinone (for 10mM should measure 17.415mg in 10mL)
# 10uM anthraquinone-2-sulfonate (for 10mM should measure 31.026mg in 10mL)
# 2uM benzyl viologen (1,1'-Dibenzyl-4,4'bipyridinium Dichloride Hydrate) (for 2mM should measure 8.187mg)
# 1uM phenylsafranine (for 1mM should measure 3.228mg)
# 1uM indigo-carmine (for 1mM should measure 4.66mg)
 
Our HRP concentration will be roughly 30uM (in order to better observe the Q-bands). Also, the cuvette path length is shorter than 1cm, so we'll need a higher concentration to observe spectral changes.
 
The final concentration of DTT should be 2000X the HRP concentration. This comes out to 60mM. We should perform 1uL additions. The initial volume in the cuvette will be 1.1mL (1mL is initially added to the cuvette and 0.1mL used to fill the honeycomb). (the sodium dithionite stock solution should be ~10M unfortunately the [http://www.chemicalbook.com/ChemicalProductProperty_EN_CB1155576.htm max solubility] is 1.4M).
 
 
==Data==
 
The buffer that was made yesterday will be used today with more degassing.
 
<u>Sodium Dithionite</u>
 
6.3419g in 25.0mL --> 1.46M
 
Degassed for 2 hours
 
<u>Dyes</u>
 
All of the dyes were placed in the same 10mL volumetric
 
# Duroquinone: 33.3mg in 10mL --> 20.3mM
# 2-hydroxy-1,4-naphthoquinone 18.0mg in 10mL --> 10.3mM
# anthraquinone-2-sulfonate 30.8mg --> 9.93mM
# benzyl viologen 8.7mg --> 2.1mM
# phenylsafranine 2.6mg --> 0.81mM
# indigo-carmine 4.6mg --> 0.99mM
 
 
These dyes were diluted by a factor of 1000 (25uL into 25mL). This solution was then degassed for 1 hour.
 
[[Image:MRH2013-09-18 10.48.07.jpg|500px]]
 
The dye solution has a blue-ish tint to it.
 
<u>HRP</u>
 
Measured 13.2mg in 10mL (dye solution) --> 33uM
 


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.




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Objective

We're going to perform a redox titration on HRP in order to determine the standard potential of this protein.

Description

We will use a Pine Instruments Honeycomb Spectroelectrochemical cell coupled to a WaveNow USB Potentiostat. The redox reaction will be monitored using a galvanic cell setting. UV Vis spectra will be recorded on an Ocean Optics Jaz Spectrometer. We will specifically use the Q-band to observe redox state following with my results from yesterday.

Following the procedure in this reference, we will add DTT in 1uL increments and observe both the UVVis spectrum and the open circuit potential of the SpecEchem cell. We will ultimately plot %oxidized or %reduced versus voltage read.

The degassed buffer will contain:

  1. 50mM Tris
  2. 50mM NaCl

and the following redox mediators (in order to stabilize the solution potential)

  1. 20uM duroquinone (tetramethyl-1,4-benzoquinone) (for 20mM should measure 32.8mg in 10mL)
  2. 10uM pyocyanine (we didn't receive this chemical)
  3. 10uM 2-hydroxy-1,4-naphthoquinone (for 10mM should measure 17.415mg in 10mL)
  4. 10uM anthraquinone-2-sulfonate (for 10mM should measure 31.026mg in 10mL)
  5. 2uM benzyl viologen (1,1'-Dibenzyl-4,4'bipyridinium Dichloride Hydrate) (for 2mM should measure 8.187mg)
  6. 1uM phenylsafranine (for 1mM should measure 3.228mg)
  7. 1uM indigo-carmine (for 1mM should measure 4.66mg)

Our HRP concentration will be roughly 30uM (in order to better observe the Q-bands). Also, the cuvette path length is shorter than 1cm, so we'll need a higher concentration to observe spectral changes.

The final concentration of DTT should be 2000X the HRP concentration. This comes out to 60mM. We should perform 1uL additions. The initial volume in the cuvette will be 1.1mL (1mL is initially added to the cuvette and 0.1mL used to fill the honeycomb). (the sodium dithionite stock solution should be ~10M unfortunately the max solubility is 1.4M).


Data

The buffer that was made yesterday will be used today with more degassing.

Sodium Dithionite

6.3419g in 25.0mL --> 1.46M

Degassed for 2 hours

Dyes

All of the dyes were placed in the same 10mL volumetric

  1. Duroquinone: 33.3mg in 10mL --> 20.3mM
  2. 2-hydroxy-1,4-naphthoquinone 18.0mg in 10mL --> 10.3mM
  3. anthraquinone-2-sulfonate 30.8mg --> 9.93mM
  4. benzyl viologen 8.7mg --> 2.1mM
  5. phenylsafranine 2.6mg --> 0.81mM
  6. indigo-carmine 4.6mg --> 0.99mM


These dyes were diluted by a factor of 1000 (25uL into 25mL). This solution was then degassed for 1 hour.

The dye solution has a blue-ish tint to it.

HRP

Measured 13.2mg in 10mL (dye solution) --> 33uM




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