User:Khyra A. Neal/Notebook/Chem 571/2014/10/15

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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 NOTE The observed dialysis solution of 50 mM CaCl2 was colorless and the colloid precipitated out. Free Lysozyme is detected at 50 mM CaCl2 because the colloid precipitated out of solution. 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 Dialyzed KI Samples extracted on October 14, 2014 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.