User:Jacob Esenther/Notebook/Chem 571/2014/09/23

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Objective

  • Utilize SDS-Page electrophoresis to observe protein sample solutions
  • Redo the Bradford Assay calibration curve with Lysozyme stock

Description

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

  1. The Gel was previously prepared and the Electrophoresis Cell was assembled
    1. Remove comb and tape from the gels
    2. Rinse the wells with running buffer
      1. Added 200mL 10x SDS-Page running buffer to 1800mL of H2O
    3. Assembled the electrophoresis cell (note diagrams in manual)
    4. Filled the inner and outer buffer chambers with running buffer
  2. Prepare and Load Samples
    1. Samples were prepped 09/17
    2. Samples were heated for 10 minutes at 100°C in the thermocycler
    3. Loaded 20uL of protein ladder into column 1 of the gel
    4. Loaded 20uL of samples into the appropriate lane of the gel


  1. Performed electrophoresis
    1. Ran for 30 minutes at 200V
  2. Developed/Stained the gel
    1. Placed gel in Fixative Solution (40% methanol, 10% acetic acid, 50% water) for 30 minutes
    2. Placed gel in Stain Solution (0.025% (w/v) Coomassie Blue, 10% acetic acid, 90% water) for 1 hour
    3. Placed gel in Destain Solution (10% acetic acid, 90% water) for 15 minutes
      1. Repeat this step with fresh destain solution 2 more times
  1. Bradford Assay of the Lysozyme Stock
    1. 0.50350g of Lysozyme was added to 1L of 0.90378% saline solution
    2. Tris-NaCl buffer prepared with 1.5145g Tris,0.7291g NaCl, and 250mL H2O


Data

  • Well Number
    • 1 - Protein Ladder
    • 4 - BSA Stock Solution
    • 6 - Soy Stock Solution
    • 8 - Lysozyme Colloid
    • 10 - Lysozyme Stock Solution
    • 12 - BSA Colloid


  • Bradford
Dilution # ' Initial Lysosyme (μg/mL) Bradford (μL) Bradford (μL) Final Lysozyme (μg/mL)
1 503.500 800 200 402.800
2 251.750 800 200 201.400
3 125.875 800 200 100.700
4 62.938 800 200 50.350
5 31.469 800 200 25.175
6 15.734 800 200 12.588
7 7.867 800 200 6.294
8 3.934 800 200 3.147
9 1.967 800 200 1.573

Notes

  • Bradford Assay is only effective from 1μg/mL to 10μg/mL
  • The tape was not removed from the electrophoresis, so the gel was not successful. This data will be borrowed from Monika and Paul.