User:Nancy T. Miles/Notebook/N.Miles Lab 2013-09-03/2013/09/17

From OpenWetWare

Jump to: navigation, search
Biomaterials Design Lab Main project page
Previous entry      Next entry

Objective

To determine the amount of reagent required to fully oxidize, using potassium ferricyanide, or reduce, using sodium hithionite horseradish peroxidase

Procedure

The following procedures for this experiment were obtained from User:Matt_Hartings/Notebook/AU_Biomaterials_Design_Lab/2013/09/17

oxidation

  1. Take a spectrum of the buffer we are using
  2. Make a 1mL HRP solution in your cuvette with a final concentration of between 5 and 10uM
    1. Allow this sample to diffuse over 5 minutes
    2. Note gently add all reagents together (slowly pipette) in order to reduce the amount of oxygen added to the system
    3. Take a spectrum of this sample
  3. Add 1uL of the K3[Fe(CN)6] solution to your cuvette with HRP
    1. Allow 5 minutes for the sample to equilibrate
    2. Take a spectrum
  4. Repeat the previous step until the HRP spectrum is unchanged from the time before
    1. Note - It may take a few additions before you see any change from the original
    2. Note - the best place to note the change is from the Soret peak, i.e. the absorption feature near 400nm. Upon oxidation, this peak should diminish and shift to lower wavelengths
    3. Note - In order to best monitor changes, it will be best to input your data promptly into excel to monitor changes. You will also want to normalize each spectrum to concentration (divide by the concentration). (Upon each addition of K3[Fe(CN)6], you will be changing your HRP concentration.
  5. Note the amount of K3[Fe(CN)6] required to fully oxidize your sample. Determine the ratio of concentration of K3[Fe(CN)6]/HRP in the fully oxidized sample.

reduction

Follow along with the procedure for oxidation and, instead, use sodium dithionite for the reduction. Upon reduction the Soret peak will increase in intensity and shift to higher wavelengths.

In order to prepare for tomorrow and have a better understanding of what we're doing today see the following references.

This reference highlights some of the changes that we'll be observing in the spectra. Note, specifically, figure 4. We won't be using this exact experimental technique, tho.

This reference goes more into detail into the kind of experiment we will be performing tomorrow. Note specifically the Redox Titrations portion of the Materials and Methods section.

Data

The following data are from the experiment conducted by Dr. Hartings

Buffer

3.0533g Tris and 1.4677g NaCl in 500mL water. pH set to 7.5 with 3M HCl

Final concentration: 50.4mM Tris 50.2mM NaCl

Buffer degassed by bubbling nitrogen through it for 3 hours

Sodium Dithionite

19.8mg in 10mL degassed buffer --> 11.3mM

2.5mL of this solution was diluted to 25mL with degassed buffer to make a final stock concentration of 1.13mM. This solution was degassed an extra 30 minutes.

K3[Fe(CN)6]

35.1mg in 10mL degassed buffer --> 10.7mM

2.5mL of this solution was diluted to 25mL with degassed buffer to make a final stock concentration of 1.07mM. This solution was degassed an extra 30 minutes.

Horseradish Peroxidase

6.9mg of HRP in 10.0mL degassed buffer --> 17uM

After the student experiments, we observed that there were no changes taking place, I went back and took a couple of spectra on my own.

Samples

  1. 250uL of HRP stock solution and 750uL of concentrated (~10mM) Dithionite stock solution
  2. 500uL of HRP stock solution and 500uL of concentrated (~10mM) Dithionite stock solution
  3. 750uL of HRP stock solution and 250uL of concentrated (~10mM) Dithionite stock solution
  4. 250uL of HRP stock solution and 750uL of concentrated (~10mM) K3[Fe(CN)4] stock solution

Here are the spectra


The following data are from the experiment conducted by the the group members:


Personal tools