User:Khyra A. Neal/Notebook/Chem 571/2014/09/23

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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
    • NaCl
    • Glycine
    • 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.

Image:DialysisSoakDiffSpec.jpg





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