Tasks for October 14
- Continuation of dialysis
- Preparation of 30:1 colloid solution vs CaCl2 using MWCO 3500 dialysis
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:
- 5 μM CaCl2. Opposite to it 30:1 Colloid
- 50 μM CaCl2. Opposite to it 30:1 Colloid
- 500 μM CaCl2. Opposite to it 30:1 Colloid
- 5 mM CaCl2. Opposite to it 30:1 Colloid
- 50 mM CaCl2. Opposite to it 30:1 Colloid
- Inserted screws to prevent evaporation
- Placed on low speed shaker for 1 week
- 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
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:
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)
- 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)
||mV measurement (mV)
| 5μM || 31.8
| 50μM || 40.6
| 500μM || 40.1
| 5mM || 56.6
| 50mM || 78.5
- 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