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
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Well 2
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Well 3
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Well 4
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Well 5
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0.6 g/L lysozyme
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0.6 g/L lysozyme
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0.6 g/L lysozyme
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0.6 g/L lysozyme
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0.6 g/L lysozome
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2 mM KI
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5 mM KI
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10 mM KI
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25 mM KI
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50 mM KI
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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)
|
Stock |
0.121 |
1.169902913
|
2 mM |
0.115 |
0.878640777
|
5 mK |
0.124 |
1.315533981
|
10 mM |
0.114 |
0.830097087
|
25 mM |
0.111 |
0.684466019
|
50 mM |
0.115 |
0.878640777
|
|
Fluorescence Results
UV-VIS Results
Primary Experiment Dialysis #2 Set-up
30:1 colloid solution vs CaCl2 using MWCO 3500
Well 1
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Well 2
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Well 3
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Well 4
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Well 5
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30:1 colloid
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30:1 colloid
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30:1 colloid
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30:1 colloid
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30:1 colloid
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50 mM CaCl2
|
5 mM CaCl2
|
0.5 mM CaCl2
|
50 μM CaCl2
|
5 μM CaCl2
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