User:Moira M. Esson/Notebook/CHEM-571/2013/09/17: Difference between revisions
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*Added .419mL of buffer to reach a volume of 1mL. | *Added .419mL of buffer to reach a volume of 1mL. | ||
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==UV/vis== | ==UV/vis== | ||
Figure 1. Normalized Absorption Spectra of the Oxidation of HRP with K<sub>3</sub>[Fe(CN)<sub>6</sub>] | Figure 1. Normalized Absorption Spectra of the Oxidation of HRP with K<sub>3</sub>[Fe(CN)<sub>6</sub>] | ||
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[[Image: | [[Image:NormalizedabsoxidatHRP09172013zem.png|500px]] | ||
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Figure 2. Enlarged Absorption Spectra of the Oxidation of HRP with K<sub>3</sub>[Fe(CN)<sub>6</sub>] | |||
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[[Image:ZoomedinnormalizedabsoxidationHRPzem.png|500px]] | |||
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*Initial drop in absorbance occurred after the addition of 2uL K<sub>3</sub>[Fe(CN)<sub>6</sub>]. | |||
*The addition of 2uL of K<sub>3</sub>[Fe(CN)<sub>6</sub>] caused the complete oxidation of HRP. This indicates that HRP was mostly oxidized prior to additon of K<sub>3</sub>[Fe(CN)<sub>6</sub>]. | |||
==HRP Reduction== | |||
General Protocol: | |||
#Run UV-vis of a blank buffer sample. | |||
#Prepare a 1mL sample of 10μM HRP in 50Tris, 50Nacl, pH=7.5 buffer. This buffer should be degassed) directly in a clean glass cuvette. | |||
#Take spectrum of the prepared 10μM HRP sample. | |||
#Add 2μL of Na<sub>2</sub>S<sub>2</sub>O<sub>4</sub> to the prepared sample. | |||
#Take UV/vis spectrum of the sample. | |||
#Repeat steps 4-5 until complete oxidation of HRP. | |||
#*Note: When preparing the sample add all components '''very'' slowly | |||
#*Note: Allow all samples to sit for approximately 5 minutes before taking the spectrum | |||
#*Note: No more than 16uL should be added of the Na<sub>2</sub>S<sub>2</sub>O<sub>4</sub> to the sample. | |||
<br> | <br> | ||
==UV/vis== | |||
Figure 1. Normalized Absorbance spectrum of the reduction of HRP with sodium Na<sub>2</sub>S<sub>2</sub>O<sub>4</sub> | |||
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[[Image:NormalizedreductionHRPzem09172013.png|500px]] | |||
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*Note: Only 10uL were added because there were noted discrepencies in the group data. Reduction of HRP will be repeated next lab. | |||
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Revision as of 11:15, 18 September 2013
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Objectives
HRP OxidationGeneral Protocol:
UV/visFigure 1. Normalized Absorption Spectra of the Oxidation of HRP with K3[Fe(CN)6]
HRP ReductionGeneral Protocol:
UV/visFigure 1. Normalized Absorbance spectrum of the reduction of HRP with sodium Na2S2O4
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