Task for September 2014
- To run electrophoresis
- To prepare SDS-PAGE
- To run SDS-PAGE
Procedure followed as described by Dr. Hartings on Sept. 25 2013
Electrophoresis
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
Note: 200mL of running buffer (10x SDS-PAGE running buffer 30.3 g Tris, 144.1g Clycine, 10g SDS, water to 1L) were diluted to 2 L. This amount was necessary in order to fill up the inner and outer buffer chambers
Prepare and Load Samples
- Five samples in centrifuge tubes were prepared on Sept. 17 2014.
- They were as follows: BSA, lysozyme, [Au]:[BSA]=30:1, [Au]:[Lysozyme]=30:1, and soy protein. Refer to lab manual from last week for detailed information on how they were made.
- Heat samples for 5 minutes at 100C (in the thermocycler)
- Load 15μL of protein ladder into column 11 of your gel
- Load 15μL of your samples into the appropriate lane of your gel
- Well 1: BSA
- Well 3: Lysozyme
- Well 5: [Au]:[BSA]=30:1 colloid
- Well 7: [Au]:[Lysozyme]=30:1 colloid
- Well 9: Soy
- Well 11: Protein ladder (Precision Plus Protein All Blue standards)
- Perform electrophoresis
- Run for 30 minutes at 200V
Perform Electrophoresis
- Run for 30 minutes at 200V
Develop/Stain your gel
- Place gel in Fixative Solution (40% methanol, 10% acetic acid, 50% water) for 30 minutes.
- Note: our gel was placed on top of Paul group's gel
- Place gel in Stain Solution (0.025% (w/v) Coomassie Blue, 10% acetic acid, 90% water) for 1 hour
- Note: our gel was placed on the bottom of the container
- Place gel in Destain Solution (10% acetic acid, 90% water) for 15 minutes
- Repeat this step with fresh destain solution 2 more times
Bradford analysis for protein content
- Both for dialyzed and non-dyalized BSA colloid solutions
- Also for outside solutions
- 25000 g/mol MW cut-off glycine soak
- 3500 g/mol MW cut-off glycine with colloid
- 3500 g/mol MW cut-off H2O soak
- 3500 g/mol MW cut-off NaCl soak
- 3500 g/mol MW cut-off glycine soak
Note: protein seemed to hsve precipitated out of solution <br.>
Bradford analysis of outside solutions:
- 600μL of outside solution
- 200μL Bradford reagent
- 200μL Tris/NaCl buffer
Run UV-Vis from 400 to 800 nm.
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