User:Alicia Rasines Mazo/Notebook/CHEM-571 Experimental Biological Chemistry/2014/10/14

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Contents

Tasks for October 14

  • SDS-PAGE
  • Continuation of dialysis
  • Preparation of 30:1 colloid solution vs CaCl2 using MWCO 3500 dialysis

SDS-PAGE #2

Procedures as listed by Dr. Fox on Oct. 14

  • 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
  • One group prepare additional 1 L of running buffer from 10X concentrate


Note: Ideal concentration for pure protein is 0.5 - 4.0 μg (40 - 60 μg for crude samples).
Using 10 μL of 0.12 g/L will load 1.2 μg protein into the well if entire 20 μL is used.

Preparation of 30:1 colloid solution vs CaCl2 using MWCO 3500 dialysis

A new dialysis experiment was prepared using 3,500 MWCO tubing.
The wells were matched up in the following way. Note that 1 mL of the following were added to each well:

  1. 5 μM CaCl2. Opposite to it 30:1 Colloid
  2. 50 μM CaCl2. Opposite to it 30:1 Colloid
  3. 500 μM CaCl2. Opposite to it 30:1 Colloid
  4. 5 mM CaCl2. Opposite to it 30:1 Colloid
  5. 50 mM CaCl2. Opposite to it 30:1 Colloid
  • Inserted screws to prevent evaporation
  • Placed on low speed shaker for 1 week

Dialysis Data

Bradford Analysis

  • Only running bradford analysis of protein-containing solutions, that is Colloid 1, 2, 3, 4 and 5, since the protein cannot diffuse through 3500 MWCO.
  • Remove 100 μL of solution from each chamber (7 in all) and run Bradford analysis
    • Bradford reagent should be diluted 1:3 with 50mM Tris/50mM NaCl
    • Recall, 100 μL solution + 200 μL diluted Bradford + 700 μL Tris/NaCl buffer
    • PS cuvettes, measuring 400 - 800 nm

Image:Bradford Colloid 141014.png

Bradford Calibration curve

  • Did Bradford analysis for undialysed lysozyme solutions with concentrations 0.12, 0.3, 0.6 and 1 g/L.
  • The calibration curve below:

Image:Lysozyme-Bradford CV.png
UV-Vis Absorption
Transfer 50 μL to a small volume UV cuvette & measure UV absorption

    • Be sure your cuvette is clean before hand. Use SDS, HCl, HPLC, & methanol washes
    • Measure entire 200 - 400 nm range. (Care about peak at 280 nm. Need to compare to fluorescence data to see how the binding is affected)

Fluorescence

  • Measured fluorescence of dyalised solutions
  • Fluorescence (glass microcuvette & 100 fold diluted) for all protein solutions
    • 10μL of lysozyme were diluted to 1mL by adding HPLC. (Remember to account for dilution factor later on)

Ca2+ ISE

CaCl2 concentration mV measurement (mV)
5μM 31.8
50μM 40.6
500μM 40.1
5mM 56.6
50mM 78.5

Buffer Preparation

  • Prepared 50 mL 66 mM potassium phosphate buffer, pH 6.319
    • Use HPLC water
    • 8.98 mg/mL KH2PO4 should produce pH 4 - 4.1
    • Add 1 M KOH to adjust pH to 6.24. To avoid this step a mixture of monobasic and dibasic potassium phosphate was used.
      • 0.00265 mol × 136.09 g/mol = 0.361 g monobasic KH2PO4
      • 0.0006 mol × 174.18g/mol = 0.105 g dibasic KH2PO4
    • Store
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