User:Daniel-Mario Larco/Notebook/AU Biodesign Lab - 09/03/2013/2014/04/15

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Objective

  • Test HRP pellet
  • Spin myoglobin and HB to see if it breaks apart
  • UV/Vis myoglobin and Hb, spin, test again

Procedure

  1. Make 0.1g/mol solutions of all three proteins in 50mM Tris buffer
  2. Spin solutions at 19000 rpm for 3 hours
  3. Test for HRP activity in the pellets
    1. Make OPD solution (1mg/mL)
    2. Make H2O2 solution
      1. 113uL 30% H2O2 in 887uL H2O
    3. Make mixture to be tested
      1. 2.025mL OPD solution
      2. 0.75mL H2O2 solution
      3. Take UV-Vis
      4. Add 0.225mL

Data

Image:Screen Shot 2014-04-16 at 5.46.16 PM.png UV-Vis of Hemoglobin solution before Spin

Image:Screen Shot 2014-04-16 at 5.46.24 PM.png UV-Vis of Myoglobin solution before Spin

Image:Screen Shot 2014-04-16 at 5.47.09 PM.png UV-Vis of AuNP solution before spin

  • Used for concentration calculations and size

Image:Screen Shot 2014-04-16 at 5.47.02 PM.png

Image:Screen Shot 2014-04-16 at 5.46.34 PM.png

Image:Screen Shot 2014-04-16 at 5.46.45 PM.png

Image:Screen Shot 2014-04-16 at 5.46.53 PM.png The last three images represent the activity of the pellets of HRP:AuNP conjugates for the three methods

Notes

When the tubes were removed from the centrifuge, none of the solutions had separated, pelleted out. There was a homogenous mixture for all three. The tubes were stored in the fridge over night to test for activity.


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