Tasks
SDS Page
- 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
Analysis of dialyzed from october 8
- Bradford Analysis
- Remove 20 μL of each solution + add 200 μL of 1:3 diluted bradford (with 50mM Tris/50mM NaCl)+ further dilute to 1 mL with 50 mM Tris/NaCl
- Measure UV-Vis from 400 nm - 800 nm (following usual protocol: run blank of Tris/NaCl buffer + undialyzed 0.6 g/L Lysozyme solution with Bradford reagent)
- Fluorescence of lysozyme containing solutions
- 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
- UV-Vis
- Run Uv-Vis on protein containing solutions that were dialyzed on October 8, 2014
- Run from 200 nm -400 nm
- Ca2+ ISE
- measured all the samples originally containing 'CaCl2
| CaCl2 concentration
|
mV measurement (mV)
|
| 5μM |
31.8
|
| 50μM |
40.6
|
| 500μM |
40.1
|
| 5mM |
56.6
|
| 50mM |
78.5
|
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
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
Prepare Calibration curve for Lysozyme
- Bradford analysis for undialysed lysozyme solutions with concentrations 0.12, 0.3, 0.6 and 1 g/L, as described above.
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