User:Matt Hartings/Notebook/AU Biomaterials Design Lab/2013/09/18: Difference between revisions
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==Description== | ==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]. 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]]. | 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 | 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 | |||
# 100mM NaCl | |||
and the following redox mediators (in order to stabilize the solution potential) | |||
# 20uM duroquinone | |||
# 10uM pyocyanine | |||
# 10uM 2-hydroxy-1,4-naphthoquinone | |||
# 10uM anthraquinone-2-sulfonate | |||
# 2uM benzyl viologen | |||
# 1uM phenylsafranine | |||
# 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. | |||
==Data== | ==Data== | ||
Revision as of 19:16, 17 September 2013
 Biomaterials Design Lab
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ObjectiveWe're going to perform a redox titration on HRP in order to determine the standard potential of this protein. DescriptionWe 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:
and the following redox mediators (in order to stabilize the solution potential)
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. Data
NotesThis area is for any observations or conclusions that you would like to note.
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