User:Matt Hartings/Notebook/AU Biomaterials Design Lab/2013/09/18: Difference between revisions
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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 | 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
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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. 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
NotesThis area is for any observations or conclusions that you would like to note.
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