User:Trisha I. Ibeh/Notebook/Trisha Notebook/2013/09/17

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Template for this lab is taken from Dr. Hartings. Values are altered to accurately describe the lab that was conducted on this day. The template can be found here

Objective

To determine the amount of reagent that is required to fully oxidize or fully reduce horseradish peroxidase. For HRP oxidation, potassium ferricyanide, K3[Fe(CN)6] will be used. For HRP reduction, sodium dithionite will be used.UV-Vis spectrum will be used to monitor the redox reactions of HRP. This is being done as preparation for the next lab (9/18/13) where the redox potential of HRP will be determined.

Procedure

The manual for Bio- Rad Mini Protean system can be found here

1. Prepare the Gel and Assemble the Electrophoresis Cell

  1. Remove comb and tape from the gels
  2. Rinse the wells with running buffer
  3. Assemble the electrophoresis cell (note diagrams in manual)
  4. Fill the inner and outer buffer chambers with running buffer

2. Prepare and Load Samples

  1. You prepped your samples yesterday
  2. Heat your samples for 5 minutes at 100C (in the thermocycler)
  3. Load 20uL of protein ladder into column 1 of your gel
  4. Load 20uL of your samples into the appropriate lane of your gel

3. Perform electrophoresis

  1. Run for 30 minutes at 200V (.2Amps)

4. Develop/Stain your gel

  1. Place gel in Fixative Solution (40% methanol, 10% acetic acid, 50% water) for 30 minutes
  2. Place gel in Stain Solution (0.025% (w/v) Coomassie Blue, 10% acetic acid, 90% water) for 1 hour
  3. Place gel in Destain Solution (10% acetic acid, 90% water) for 15 minutes

1. Repeat this step with fresh destain solution 2 more times Data

Data

Notes

Lanes 1 and 3 were incorrectly aliquoted. As a result of this, there is no data (bands) displayed.

In the lane with the hemoglobin sample, the hemoglobin rose to the top of the lane.

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