User:Alicia Rasines Mazo/Notebook/CHEM-571 Experimental Biological Chemistry/2014/10/14

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Tasks for October 14 SDS-PAGE Continuation of dialysis Preparation of 30:1 colloid solution vs CaCl2 using MWCO 3500 dialysis SDS-PAGE #2 Procedures as listed by Dr. Fox on Oct. 14 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 One group prepare additional 1 L of running buffer from 10X concentrate Note: Ideal concentration for pure protein is 0.5 - 4.0 μg (40 - 60 μg for crude samples). Using 10 μL of 0.12 g/L will load 1.2 μg protein into the well if entire 20 μL is used. Preparation of 30:1 colloid solution vs CaCl2 using MWCO 3500 dialysis A new dialysis experiment was prepared using 3,500 MWCO tubing. <br.> The wells were matched up in the following way. Note that 1 mL of the following were added to each well: 5 μM CaCl2. Opposite to it 30:1 Colloid 50 μM CaCl2. Opposite to it 30:1 Colloid 500 μM CaCl2. Opposite to it 30:1 Colloid 5 mM CaCl2. Opposite to it 30:1 Colloid 50 mM CaCl2. Opposite to it 30:1 Colloid Inserted screws to prevent evaporation Placed on low speed shaker for 1 week Dialysis Data Bradford Analysis Only running bradford analysis of protein-containing solutions, that is Colloid 1, 2, 3, 4 and 5, since the protein cannot diffuse through 3500 MWCO. Remove 100 μL of solution from each chamber (7 in all) and run Bradford analysis Bradford reagent should be diluted 1:3 with 50mM Tris/50mM NaCl Recall, 100 μL solution + 200 μL diluted Bradford + 700 μL Tris/NaCl buffer PS cuvettes, measuring 400 - 800 nm Bradford Calibration curve Did Bradford analysis for undialysed lysozyme solutions with concentrations 0.12, 0.3, 0.6 and 1 g/L. The calibration curve below: <br.> UV-Vis Absorption <br.> Transfer 50 μL to a small volume UV cuvette & measure UV absorption Be sure your cuvette is clean before hand. Use SDS, HCl, HPLC, & methanol washes Measure entire 200 - 400 nm range. (Care about peak at 280 nm. Need to compare to fluorescence data to see how the binding is affected) Fluorescence Measured fluorescence of dyalised solutions Fluorescence (glass microcuvette & 100 fold diluted) for all protein solutions 10μL of lysozyme were diluted to 1mL by adding HPLC. (Remember to account for dilution factor later on) Ca2+ ISE CaCl2 concentration mV measurement (mV) 5μM 31.8 50μM 40.6 500μM 40.1 5mM 56.6 50mM 78.5 Buffer Preparation Prepared 50 mL 66 mM potassium phosphate buffer, pH 6.319 Use HPLC water 8.98 mg/mL KH2PO4 should produce pH 4 - 4.1 Add 1 M KOH to adjust pH to 6.24. To avoid this step a mixture of monobasic and dibasic potassium phosphate was used. 0.00265 mol × 136.09 g/mol = 0.361 g monobasic KH2PO4 0.0006 mol × 174.18g/mol = 0.105 g dibasic KH2PO4 Store