October 15, 2014
Tasklist
Electrophoresis
- Warm solutions for 5 minutes at 40°C that were prepared on October 14, 2014
- Load samples to premade gel
- Well 1: Precision Plus Protein All Blue Standards
- Well 3: 0.12 g/L lysozyme Unk A
- Well 4: 30:1 Lysozyme colloid
- Well 5: 0.12 g/L Lysozyme
- Well 6: 0.6 g/L Lysozyme
- Wells 8-12 contain solutions prepared by Dr. Fox
- Run for 40 minutes at 200V
- Develop/Stain your gel
- Place gel in Fixative Solution (40% methanol, 10% acetic acid, 50% water) for 30 minutes
- Place gel in Stain Solution (0.025% (w/v) Coomassie Blue, 10% acetic acid, 90% water) for 1 hour
- Place gel in Destain Solution (10% acetic acid, 90% water) for 15 minutes
- Repeat this step with fresh destain solution 2 more times
The original protocol for electrophoresis was written by Dr. Hartings
Bradford Analysis
Bradford Analysis was conducted on the dialyzed 30:1 colloid against CaCl2 solutions
- 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 30:1 colloid solution with Bradford reagent
UV-Vis and Fluorescence of Dialyzed Colloid Solutions
Fluorescence
- 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
- Clean the cuvette after each use with several SDS, HCl, HPLC, & methanol washes
UV-Vis of Colloid Solutions
- Transfer 100 μL of dialyzed solution to small volume glass cuvette
- Run Uv-Vis on protein containing solutions
Run from 200 nm -400 nm
Ca2+ ISE
ISE for protein containing Ca2+ ions
Ca2+ ISE
| CaCl2 concentration
|
mV
|
| 5μM |
31.8
|
| 50μM |
40.6
|
| 500μM |
40.1
|
| 5mM |
56.6
|
| 50mM |
78.5
|
Determination of KI Concentration by Titration
- Standardization:
- AgNO3 + NH4SCN → AgSCN + NH4NO3
- KI + AgNO3(excess) → AgI (s) + AgNO3 +KNO3
- Initial AgNO3: 0.075 M AgNO3 x 0.002 L AgNO3 = 0.00015 mol AgNO3
- AgNO3 (neutralized): 0.00968 M NH4SCN x 0.0104 L NH4SCN x (1 mol AgNO3 / 1 mol NH4SCN ) = 0.000100672 mol AgNO3
- Moles of KI: 0.00015 mol AgNO3 initial - 0.000100672 mol AgNO3 neutralized = 0.0000493 mol KI
- [KI]= 0.0000493 mol KI / 0.001 L KI = 49.3 mM KI
NOTE The final concentration of NH4SCN was determined by titration standardization on October 8, 2014
Titrations were performed as follows
- 9.68 mM NH4 is being titrated against 3 mL 1M HNO3, 10 mL deionized H2O , 200 μLFerric alum indicator, and 0.5 mL of dialyzed sample
- Lysozyme against 2 mM KI
- 15.1 mL NH4SCN used
- [KI] = 7.6 mM
- Lysozyme against 5 mM KI
- 14.2 mL NH4SCN used
- [KI] = 25 mM
- Lysozyme against 10 mM KI
- 14.0 mL NH4SCN used
- [KI] = 29 mM
- Lysozyme against 25 mM KI
- 13.7 mL NH4SCN used
- [KI] = 34.8 mM
- Lysozyme against 50 mM KI
- 12.5 mL NH4SCN used
- [KI] = 58 mM
- 2 mM KI against Lysozyme
- 13.6 mL NH4SCN used
- [KI] = 36.7 mM
- 5 mM KI against Lysozyme
- 13.4 mL NH4SCN used
- [KI] = 40.6 mM
- 10 mM KI against Lysozyme
- 13.2 mL NH4SCN used
- [KI] = 44.4 mM
- 25 mM KI against Lysozyme
- 12.6 mL NH4SCN used
- [KI] = 56.1 mM
- 50 mM KI against Lysozyme
- 11.9 mL NH4SCN used
- [KI] = 69.6 mM
NOTE Our values are indicative of experimental error. We found that our initial dilutions of KI (2-25 mM) were not prepared at the indicated concentrations. All diluted KI solutions were discarded and new solutions were prepared using fresh 50 mM KI on October 21, 2014.
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