User:Alicia Rasines Mazo/Notebook/CHEM-571 Experimental Biological Chemistry/2014/09/23

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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
  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

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
  1. 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.
  2. Heat samples for 5 minutes at 100C (in the thermocycler)
  3. Load 15μL of protein ladder into column 11 of your gel
  4. 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)
  5. Perform electrophoresis
  6. Run for 30 minutes at 200V
Perform Electrophoresis
  • Run for 30 minutes at 200V
Develop/Stain your gel
  1. 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
  2. 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
  3. 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:

  1. 600μL of outside solution
  2. 200μL Bradford reagent
  3. 200μL Tris/NaCl buffer

Run UV-Vis from 400 to 800 nm.