User:Matt Hartings/Notebook/AU Biomaterials Design Lab/2016/01/13

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Objective

The referees of the microgel paper wanted me to determine if the protein hydrolyzes during the reaction for making hydrogels. So, I'm going to run 2 reaction (one with iron and one without iron) to see if the protein is degraded by the KOH and KNO3.

Description

Following Puja's methods

  1. Heat a water bath to 80C
  2. Degas water for making FeSO4 solution
  3. Add the following to a falcon tube:
    1. 1.87 mL of 1M KOH
    2. 8.889 mL of 0.9M KNO3
    3. 2.338 mL of 15.4 uM Hemoglobin
    4. 4.903 mL of water
  4. Prepare the FeSO4 solution using the degassed water to prevent oxidation of Fe before it goes into the reaction
    1. 2.085 g FeSO4 (MW 278.01 g/mol)
    2. 25 mL degassed water
  5. Place tube around mixing shaft
  6. Place water bath around tube
  7. Start mixing at 2500 rpm
  8. Add 2 mL of FeSO4 solution
  9. Let react for 90 minutes and place in freezer overnight

Now I need to make a reaction that has no FeSO4 to look at the effects of just KOH and KNO3

  1. Heat a water bath to 80C
  2. Add the following to a falcon tube:
    1. 1.87 mL of 1M KOH
    2. 8.889 mL of 0.9M KNO3
    3. 2.338 mL of 15.4 uM Hemoglobin
    4. 6.903 mL of water
  3. Place tube around mixing shaft
  4. Place water bath around tube
  5. Start mixing at 2500 rpm
  6. Add 2 mL of FeSO4 solution
  7. Let react for 90 minutes and place in freezer overnight

I also need to prep solutions to run electrophoresis

  1. 10X running buffer
    1. 30.3 g Tris
    2. 144.1 g glycine
    3. 10 g SDS
    4. Add water to 1L
    5. stir on stir plate overnight to mix solution
  2. 2X sample buffer
    1. 3.75 mL of 0.5 M Tris (pH 6.8)
    2. 7.5 mL of glycerol
    3. 0.3 mL of 1% bromophenol blue
    4. 0.6 g SDS
    5. H2O to 30 mL

Data

  • Add data and results here...

Notes

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