User:Beatriz Gimenez De C./Notebook/571/2014/10/14

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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 October 8 dialysis analysis 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. Run fluorescence from 300 nm -550 nm and excitation at 280 nm UV-Vis Run Uv-Vis on protein containing solutions obtained from dialisis 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 Added 0.361 g of monobasic potassium phosphate + 0.105 g of dibasic potassium phosphate to 50 mL volumetric flask + filled with HPLC water Final pH: 6.319 Prepare New Dialysis for 30:1 Colloid vs CaCl2 Use 3500 MWCO tubing Add 30:1 colloid solution to 5 cells Add 5 μM, 50 μM, 500 μM, 5 mM, and 50 mM CaCl2 to the opposite cells Follow usual protocol 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.