September 23, 2014
SDS-Page and Electrophoresis
The protocol for todays tasks can be found here Electrophoresis
- Prepare SDS-Page Running Buffer
- 10X SDS-Page Running Buffer needs to be diluted by a factor of 10.
- Preparing 2 L →200 mL SDS-Page Running Buffer in 1800 mL of H2O
- Heat samples prepared on September 17,2014 for 5 minutes at 100°C using the thermocycler.
- Prep Gel and Assemble the Electrophoresis
- Load 15 μL of samples that were prepared on September 17,2014.
- Well 1: BSA
- Well 3: Lysozyme
- Well 5: BSA Colloid
- Well 7: Lysozyme Colloid
- Well 9: Soy
- Well 11: All Blue Protein Standard (provided by Bio-Rad)
- Run Electrophoresis for 30 minutes at 200 V
- Develop/Stain 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 with fresh destain solution twice
Analysis of Dialyzed Solutions from September 16,2014
Bradford Analysis of Soak Solutions Outside of Tubing
We are conducting a Bradford Assay on the outside soak solutions from the dialysis. The solutions are as follows:
- 3500 g/mol MWCO tubing Soak Solutions
- Pure Soak Water (HPLC Water)
- 25k g/mol MWCO tubing in Glycine soak
- In a 1.5 mL centrifuge tube add:
- 600 μL of Soak Solution
- 200 μL Bradford Reagent (diluted 1:4)
- 200 μL Tris/ NaCl Buffer (prepared on September 16,2014 to bring total volume to 1 mL
- Close centrifuge tube, vortex for 5 seconds, and let stand for 5 minutes.
- Run UV-Vis between 400 nm and 800 nm.