User:Monika Gasiorek/Notebook/CHEM-571 2014F/2014/10/14

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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 Well 2 Well 3 Well 4 Well 5 0.6 g/L lysozyme 0.6 g/L lysozyme 0.6 g/L lysozyme 0.6 g/L lysozyme 0.6 g/L lysozome 2 mM KI 5 mM KI 10 mM KI 25 mM KI 50 mM KI 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 Well 2 Well 3 Well 4 Well 5 30:1 colloid 30:1 colloid 30:1 colloid 30:1 colloid 30:1 colloid 50 mM CaCl2 5 mM CaCl2 0.5 mM CaCl2 50 μM CaCl2 5 μM CaCl2