To run SDS-PAGE to observe amount of hemoglobin remaining after yesterday's digestion. This should be a complimentary measurement to our UV-Vis analysis from yesterday.
We will be using a Bio-Rad Mini Protean system with pre-cast Mini Protean TGX gels. The manual for this system can be found here We will be running SDS-PAGE followed by gel development with Coomassie Blue staining.
Below is a short description of how we will proceed. Please refer to the manual for more detailed instructions
- Prepare the Gel and Assemble the Electrophoresis Cell
- Remove comb and tape from the gels
- Rinse the wells with running buffer
- Assemble the electrophoresis cell (note diagrams in manual)
- Fill the inner and outer buffer chambers with running buffer
- Prepare and Load Samples
- You prepped your samples yesterday
- Heat your samples for 5 minutes at 100C (in the thermocycler)
- Load 20uL of protein ladder into column 1 of your gel
- Load 20uL of your samples into the appropriate lane of your gel
- Perform electrophoresis
- Run for 30 minutes at 200V (I need to make sure our power source can do this)
- Develop/Stain your gel
- Place gel in Fixative Solution (40% methanol, 10% acetic acid, 50% water) for 30 minutes
- Place gel in Stain Solution (0.025% (w/v) Coomassie Blue, 10% acetic acid, 90% water) for 1 hour
- Place gel in Destain Solution (10% acetic acid, 90% water) for 15 minutes
- Repeat this step with fresh destain solution 2 more times
- Add data and results here...
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