User:Monika Gasiorek/Notebook/CHEM-571 2014F/2014/10/14

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October 14, 2014

Preparation for SDS-PAGE - Take 2

  • 10 μL of 0.6 g/L lysozyme was mixed with 10μL SDS-PAGE sample buffer (30.3 g Tris, 144.1g Clycine, 10g SDS in 1 L water -->diluted by a factor of 10) in a 1.5 mL centrifuge vial
  • 10 μL of 0.12 g/L lysozyme was mixed with 10μL SDS-PAGE sample buffer in a 1.5 mL centrifuge vial
  • 10 μL of 30:1 Au/lysozyme colloid was mixed with 10μL SDS-PAGE sample buffer in a 1.5 mL centrifuge vial
  • 10 μL of 0.12 g/L unknown protein was mixed with 10 μL SDS-PAGE sample buffer in a 1.5 mL centrifuge vial
  • The four samples were placed in a heating block (set at 90 °C) for 5 minutes and stored in the fridge overnight


  • 50 mL of a 66 mM Potassium Phosphate buffer with pH 6.24 was prepared using HPLC water, monobasic and dibasic potassium phosphate solids, KHPO4 and K2HPO4

Primary Experiment Dialysis #1 Work-up

0.6 g/L Lysozyme vs KI using MWCO 3500

Well 1 Well 2 Well 3 Well 4 Well 5
0.6 g/L lysozyme 0.6 g/L lysozyme 0.6 g/L lysozyme 0.6 g/L lysozyme 0.6 g/L lysozome
2 mM KI 5 mM KI 10 mM KI 25 mM KI 50 mM KI

Necessary Analyses:

  • Bradford - completed
    • 5 lysozyme samples, 0.6 g/L lysozyme reference, and Bradford blank
    • 20 μL sample, 200 μL 1:4 Bradford, 780 μL 50 mM Tris/50 mM NaCl
  • Fluorescence - completed
    • 5 lysozyme samples, 0.6 g/L lysozyme reference
    • 10 μL of sample in 990 μL of DI water
  • UV-Vis - completed
    • 5 lysozyme samples, 0.6 g/L lysozyme reference
    • 10 μL of sample in 990 μL of DI water
  • KI - completed; see 10/21 for results
    • 10 samples for titration with NH4SCN

Bradford Results

Wavelength Abs at 577 nm Concentration (ug/mL)
Stock0.1211.169902913
2 mM0.1150.878640777
5 mK0.1241.315533981
10 mM0.1140.830097087
25 mM0.1110.684466019
50 mM0.1150.878640777

Fluorescence Results

UV-VIS Results

Primary Experiment Dialysis #2 Set-up

30:1 colloid solution vs CaCl2 using MWCO 3500

Well 1 Well 2 Well 3 Well 4 Well 5
30:1 colloid 30:1 colloid 30:1 colloid 30:1 colloid 30:1 colloid
50 mM CaCl2 5 mM CaCl2 0.5 mM CaCl2 50 μM CaCl2 5 μM CaCl2


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