User:Alicia Rasines Mazo/Notebook/CHEM-571 Experimental Biological Chemistry/2014/10/01

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Tasks for October 1 Analysis of Dialysis Solutions from Sept. 30 Analysis of Dialysis Solution Procedure followed as detailed by Dr. Fox on Oct.1 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 Recall, 20 μL solution + 200 μL diluted Bradford + 780 μL Tris/NaCl buffer PS cuvettes, measuring 400 - 800 nm Don't forget to run a blank with just Bradford & buffer Don't forget to run your undialyzed Lysozyme stock Transfer remainder of each cell into 20 mL extraction vials Measure Ca2+ using ISE Substance mV measurement (mV) 50mM CaCl2 (4) 80.8 Lysozyme (4) 80.3 500μM CaCl2 (3) 28.8 Lysozyme (3) 28.1 Transfer 100 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 - 800 nm range Transfer 100 μL to a small volume fluorescence cuvette & measure fluorescence Be sure your cuvette is clean before hand. Use SDS, HCl, HPLC, & methanol washes excitation at 280 nm Transfer 500 - 700 μL to a 15 mL Falcon tube. Add 2 - 3 times volume of water & measure pH You want to add minimum amount of water You typically need 1.6 - 2 mL in falcon tube to cover pH probe So, if you have only 500 μL left, you'll need a 4x dilution (adding 1.5 mL water) Or, if you have 700 μL left, you only need a 3x dilution (adding 1.4 mL water) Preparation of new dialysis A new dialysis experiment was prepared using 22,000 MWCO tubing instead of 3500MWCO. <br.> The wells were matched up in the following way. Note that 1 mL of the following were added to each well: 50 mM CaCl2. Opposite to it Colloid 1 500 μM CaCl2. Opposite to it Colloid 2 HPLC water. Opposite to it Lysozyme 3 0.25 mM HCl. Opposite to it Lysozyme 4 50 mM NaCl. Opposite to it Lysozyme 5 Inserted screws to prevent evaporation Placed on low speed shaker for 1 week