Objective
- Test HRP pellet
- Spin myoglobin and HB to see if it breaks apart
- UV/Vis myoglobin and Hb, spin, test again
Procedure
- Make 0.1g/mol solutions of all three proteins in 50mM Tris buffer
- Spin solutions at 19000 rpm for 3 hours
- Test for HRP activity in the pellets
- Make OPD solution (1mg/mL)
- Make H2O2 solution
- 113uL 30% H2O2 in 887uL H2O
- Make mixture to be tested
- 2.025mL OPD solution
- 0.75mL H2O2 solution
- Take UV-Vis
- Add 0.225mL
Data
UV-Vis of Hemoglobin solution before Spin
UV-Vis of Myoglobin solution before Spin
UV-Vis of AuNP solution before spin
- Used for concentration calculations and size
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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