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:

Well 1 Well 2 Well 3 Well 4 Well 5
0.12 g/L lysozyme 0.12 g/L lysozyme 0.12 g.L lysozyme 30:1 [Au]:[Lys] colloid 30:1 [Au]:[Lys] colloid
50 μM CaCl2 50 mM CaCl2 500 μM CaCl2 50 μM CaCl2 50 mM CaCl2

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:

Solution Absorbance Concentration of Protein (µg/mL)
5uM Lysozyme 0.02 -3.733009709
5uM Lysozyme Soak 0.022 -3.63592233
50 mM Colloid 0.012 -4.121359223
50 mM Colloid Soak 0.01 -4.218446602
50 mM Lysozyme 0.035 -3.004854369
50 mM Lysozyme Soak 0.025 -3.490291262
50 uM Colloid 0.013 -4.072815534
50 uM Colloid Soak 0.023 -3.587378641
50 uM Lysozyme 0.018 -3.830097087
50 uM Lysozyme Soak 0.028 -3.344660194

[I-] by Precipitation Titration

50 mL of 50 mM KI solution was prepared and stored in a black 50 mL Falcon tube.

  • 0.41517 g of solid KI was dissolved in 50 mL of water

50 mL of 75 mM AgNO3 was prepared and stored in a black 50 mL Falcon tube.

  • 0.63788 g of solid AgNO3 was dissolved in 50 mL of water

100 mL of 10 mM NH4SCN was prepared and stored in 2 black 50 mL Falcon tubes.

  • 0.07669 g of solid NH4SCN was dissolved in 100 mL of water

NH4SCN Standardization

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.

  • Volume required to reach equivalence point: 15.60 mL

AgNO3+NH4SCN→AgSCN(s)+NH4NO3

  • Reaction is 1:1
  • 0.63788g AgNO3/50mL = 0.0127576 g/mL *2 mL *1mole/169.86 g = 0.000150204 mol AgNO3 = mol Ag = mol SCN = mole NH4SCN
  • 0.000150204 mol NH4SCN/0.0156 L

[NH4SCN]=9.63 mM

KI Standardization

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.

  • Volume required to reach equivalence point:

Trial 1: 10.15 mL Trial 2: 10.10 mL Average: 10.125 mL

KI+AgNO3→AgI+AgNO3(excess)+KNO3

KNO3+AgNO3(excess)+NH4SCN→AgSCN+NH4NO3

  • 10.125 mL (average) of NH4SCN were required to titrate excess AgNO3
    • 0.010125 L * 0.009628479 mol/L = 0.000097488 mol SCN = mol Ag
  • 2mL of original AgNO3 = 0.000150204 mol Ag
  • 0.000150204 - 0.000097488 = 0.000052716 mol Ag = mol I = mol KI
  • 1 mL of KI contains 5.2716×10-5 mol

[KI]=52.716 mM

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:

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

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.



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