User:Monika Gasiorek/Notebook/CHEM-571 2014F/2014/10/08
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October 8, 2014
Bradford Analysis of 20 kD Dialysis
Reminder of dialysis well set-up:
20 μL of each sample, 10 samples in total, were combined with 200 μL of Bradford reagent (made via previously described 1:4 preparation with 50 mM Tris/50 mM NaCl) and 780 μL of 50 mM Tris/50 mM NaCl buffer. A 200 μL Bradford/800 μL Tris/NaCl blank was run as well and subtracted from each sample's spectrum. The results are included below and include data for all 10 samples, as the 20 kD dialysis tubing was used and there was potential for free protein movement across the membrane:
NOTE: Should also have run a reference sample of the 0.12 g/L lysozyme and 30:1 colloid stock solutions for reference purposes.
The previously constructed Bradford calibration curve was utilized to determine the protein concentrations in the four lysozyme-containing cells:
[I-] by Precipitation Titration
50 mL of 50 mM KI solution was prepared and stored in a black 50 mL Falcon tube.
50 mL of 75 mM AgNO3 was prepared and stored in a black 50 mL Falcon tube.
100 mL of 10 mM NH4SCN was prepared and stored in 2 black 50 mL Falcon tubes.
3 mL of 1 M HCl, 2 mL of AgNO3, and 10 mL of water were titrated with the prepared NH4 solution in the presence of 200 μL of ferric alum indicator.
3 mL of 1 M HNO3, 1 mL of KI, and 10 mL of water were combined. 2 mL of AgNO3 was added and a precipitate was observed. The precipitate and solution were titrated with the prepared NH4 solution in the presence of 200 μL of ferric alum indicator.
Trial 1: 10.15 mL Trial 2: 10.10 mL Average: 10.125 mL
Primary Experiment Dialysis #1 Set-Up
The first dialysis of the next 6 week primary experiment was set up to run over the weekend using 3500 MWCO dialysis tubing and with the following organization:
0.6 g/L Lysozyme vs KI using MWCO 3500
NOTE: Concentrations of KI are approximate as KI standardization demonstrated a higher concentration for the 50 mM stock KI than anticipated. Because the solutions in the wells are serial dilutions of the stock, their concentrations should be adjusted accordingly.