User:Matt Hartings/Notebook/AU Biomaterials Design Lab/2013/09/18: Difference between revisions

From OpenWetWare
Jump to navigationJump to search
Line 32: Line 32:
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.
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 DTT stock solution should be ~10M).
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==
==Data==

Revision as of 05:54, 18 September 2013

Biomaterials Design Lab <html><img src="/images/9/94/Report.png" border="0" /></html> Main project page
<html><img src="/images/c/c3/Resultset_previous.png" border="0" /></html>Previous entry<html>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</html>Next entry<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html>



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
  2. 10uM pyocyanine
  3. 10uM 2-hydroxy-1,4-naphthoquinone
  4. 10uM anthraquinone-2-sulfonate
  5. 2uM benzyl viologen
  6. 1uM phenylsafranine
  7. 1uM indigo-carmine

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

  • Add data and results here...

Notes

This area is for any observations or conclusions that you would like to note.


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.




Retrieved from "https://openwetware.org/mediawiki/index.php?title=User:Matt_Hartings/Notebook/AU_Biomaterials_Design_Lab/2013/09/18&oldid=729398"