Objectives
- Find endpoints (100%) of oxidation and reduction of HRP
HRP Oxidation
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 K3[Fe(CN)6] 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 20uL should be added of the K3[Fe(CN)6] to the sample.
Table 1: Sample preparation for HRP
Dilution
|
'M1'[μM]
|
'V1' [mL]
|
'M2'[μM]
|
'V2' [mL]
|
1 |
17.25 |
.580 |
10 |
1 |
|
- Added .419mL of buffer to reach a volume of 1mL.
UV/vis
Figure 1. Normalized Absorption Spectra of the Oxidation of HRP with K3[Fe(CN)6]
Figure 2. Enlarged Absorption Spectra of the Oxidation of HRP with K3[Fe(CN)6]
- Initial drop in absorbance occurred after the addition of 2uL K3[Fe(CN)6].
- The addition of 2uL of K3[Fe(CN)6] caused the complete oxidation of HRP. This indicates that HRP was mostly oxidized prior to additon of K3[Fe(CN)6].
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 Na2S2O4 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 Na2S2O4 to the sample.
UV/vis
Figure 1. Normalized Absorbance spectrum of the reduction of HRP with sodium Na2S2O4
- 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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