User:Paul Rothenberg/Notebook/CHEM 571 Fall 2014/2014/09/23

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SDS-PAGE of Lysozyme

We are running SDS-PAGE of the samples that Monika prepared. Here is what she did:

5 samples were prepared and stored in the fridge for electrophoresis next week.

  • 20 μL of the 1g/1L lysozyme stock solution were added to a 2 mL volumetric flask. 10 μL of this dilution were added to 10 μL of a running buffer prepared by Dr. Fox
  • 20 μL of the 36.5:1 [Au]:[Lys] colloid solution were added to a 2 mL volumetric flask. 10 μL of this dilution were added to 10 μL of the running buffer
  • 20 μL of a ___:__ [Au]:[BSA] colloid solution created the first day of lab were added to a 2 mL volumetric flask. 10 μL of this dilution were added to 10 μL of the running buffer
  • 20 μL of a 1g/1L BSA stock solution were added to a 2 mL volumetric flask. 10 μL of this dilution were added to 10 μL of the running buffer
  • 0.00209 g of powdered soy protein were dissolved in a 2 mL volumetric flask. 50 μL of this stock soy solution were then added to a 5 mL volumetric flask. 10 μL of this dilution were then added to 10 μL of the running buffer


Procedure for SDS-PAGE

The protocol can be found here. The proteins solutions prepared were placed in 100°C in a thermocycler for 5 minutes to denature the protein. The running buffer was diluted 10X.

20 µL of the protein ladder was loaded, along with 20 µL of samples in each well. The ladder was in well 1, BSA in 3, BSA colloid in 5, Lysozyme in 7, Lysozyme colloid in 9, and Soy Protein in 11.

The gel was run for 30 minutes at 200V. It was fixed for 30 minutes, stained for 1 hour, and destained for 15 minutes. The gel was further rinsed by Dr. Fox.

Redo of Bradford Assay

The data was not conclusive for the Bradford assay, so we are re-running it in larger quantities with serial dilutions. 1L of 0.9% saline was prepared, and 0.50350 g of lysozyme was dissolved. Serial dilutions were prepared with the 50 mM Tris and NaCl buffer by a factor of 2 each time.

Results

Bradford UV VIS Chart.png

Bradford Calibration Graph.png