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

From OpenWetWare

(Difference between revisions)
Jump to: navigation, search
(Description)
(Description)
Line 22: Line 22:
# 50mM NaCl
# 50mM NaCl
and the following redox mediators (in order to stabilize the solution potential)
and the following redox mediators (in order to stabilize the solution potential)
-
# 20uM duroquinone
+
# 20uM duroquinone (tetramethyl-1,4-benzoquinone) (for 20mM should measure 32.8mg in 10mL)
-
# 10uM pyocyanine
+
# <strike>10uM pyocyanine</strike> (we didn't receive this chemical)
-
# 10uM 2-hydroxy-1,4-naphthoquinone
+
# 10uM 2-hydroxy-1,4-naphthoquinone (for 10mM should measure 17.415mg in 10mL)
-
# 10uM anthraquinone-2-sulfonate
+
# 10uM anthraquinone-2-sulfonate (for 10mM should measure 31.026mg in 10mL)
-
# 2uM benzyl viologen
+
# 2uM benzyl viologen (1,1'-Dibenzyl-4,4'bipyridinium Dichloride Hydrate) (for 2mM should measure 8.187mg)
-
# 1uM phenylsafranine
+
# 1uM phenylsafranine (for 1mM should measure 3.228mg)
-
# 1uM indigo-carmine
+
# 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.
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.

Revision as of 08:42, 18 September 2013

Biomaterials Design Lab Main project page
Previous entry      Next entry



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

  • 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.




Personal tools