User:Khyra A. Neal/Notebook/Chem 571/2014/10/14

Project name

Main project page Previous entry      Next entry

October 14, 2014 Tasklist 1. SDS Page #2 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 2. Bradford Analysis Analysis of Solutions that were extracted from wells that set for one week on October 8, 2014 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 0.6 g/L Lysozyme solution with Bradford reagent 3. Calibration Curve for undialyzed Lysozyme by Bradford Analysis 0.12 g/L, 0.3 g/L, 0.5 g/L, 0.6 g/L and 1 g/L Lysozyme 20 μL of Lysozyme 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. 4. 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 5. Fluorescence on protein containing solutions that were dialyzed on October 8, 2014 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 6. UV-Vis Run Uv-Vis on protein containing solutions that were dialyzed on October 8, 2014 Run from 200 nm -400 nm 7. 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