User:Khyra A. Neal/Notebook/Chem 571/2014/10/14

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October 14, 2014

Tasklist

1. SDS Page #2

  • Mix 10 μL 0.6 g/L lysozyme with 10μL SDS-PAGE sample buffer in 1.5 mL centrifuge vial
  • Mix 10 μL 0.12 g/L lysozyme with 10μL SDS-PAGE sample buffer in 1.5 mL centrifuge vial
  • Mix 10 μL 30:1 Au/lysozyme colloid with 10μL SDS-PAGE sample buffer in 1.5 mL centrifuge vial
  • Mix 10 μL 0.12 g/L unknown protein with 10 μL SDS-PAGE sample buffer in 1.5 mL centrifuge vial
  • Place in heating block (set at 90 °C) for 5 minutes
  • Store in refrigerator overnight


2. Bradford Analysis

  • Analysis of Solutions that were extracted from wells that set for one week on October 8, 2014
    • Remove 20 μL of solution from each chamber (10 in all) and run Bradford analysis
    • Bradford reagent should be diluted 1:3 with 50mM Tris/50mM NaCl
    • Dilute to 1 mL by adding 780 μL stock 50 mM Tris/NaCl
    • Measure UV-Vis from 400 nm - 800 nm using polystyrene cuvettes.
    • Run blank of Tris/NaCl buffer
    • Run UV-Vis of undialyzed 0.6 g/L Lysozyme solution with Bradford reagent

Dialyzed Lysozyme in KI.jpg

3. Calibration Curve for undialyzed Lysozyme by Bradford Analysis

  • 0.12 g/L, 0.3 g/L, 0.5 g/L, 0.6 g/L and 1 g/L Lysozyme
  • 20 μL of Lysozyme
  • Bradford reagent should be diluted 1:3 with 50mM Tris/50mM NaCl
  • Dilute to 1 mL by adding 780 μL stock 50 mM Tris/NaCl
  • Measure UV-Vis from 400 nm - 800 nm using polystyrene cuvettes.

Undialyzed Lysozyme Calibration.jpg

4. Prepare 50 mL 66 mM Potassium Phosphate Buffer

  • 0.361 g of monobasic potassium phosphate
  • 0.105 g of dibasic potassium phosphate
  • Add to 50 mL volumetric flask and dilute with HPLC water
  • Final pH is 6.319

5. Fluorescence on protein containing solutions that were dialyzed on October 8, 2014

  • Dilute Lysozyme sample 100x for each well.
  • Transfer 100 μL to a small volume fluorescence cuvette
  • Run fluorescence from 300 nm -550 nm and excitation at 280 nm

6. UV-Vis

  • Run Uv-Vis on protein containing solutions that were dialyzed on October 8, 2014
  • Run from 200 nm -400 nm

7. Prepare New Dialysis for 30:1 Colloid vs CaCl2

  • Use 3500 MWCO tubing
  • Add 1 mL 30:1 colloid solution to 5 cells on one side of chamber
  • Add 1 mL 5 μM, 50 μM, 500 μM, 5 mM, and 50 mM CaCl2 to the opposite side of the chamber (one concentration per cell)
  • Secure wells by screwing them to prevent any evaporation
  • Place on low speed shaker overnight