User:Alicia Rasines Mazo/Notebook/CHEM-571 Experimental Biological Chemistry/2014/10/08

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Tasks for October 8

  • [I-] by Titration Precipitation
  • To set up a new dialysis experiment containing [I-]

[I-] by Titration Precipitation

Procedure followed as detailed by Dr. Fox

  • Prepare 50 mL 50 mM KI Stock Solution (store in 50 mL Falcon tube)
    • 0.415g of KI were added to 50 mL volumetric flask and distilled water was added to make up to mark
  • Prepare 50 mL 75 mM AgNO3 Solution
    • Be very precise with your measurements (use Rm 205 balance)
    • 0.63701 g of AgNO3 were added to 50 mL volumetric flask and distilled water was added to make up to mark
    • Store in Black 50 mL Falcon tube
  • Prepare 100 mL 10 mM NH4SCN Stock Solution (store in Falcon tubes)
    • 0.0761 g of NH4SCN were added to 100 mL volumetric flask and distilled water was added to make up to mark
  • NH4SCN Standardization
    1. Add 3 mL 1 M HNO3 and 2 mL AgNO3 to 10 mL DI H2O
    2. Add 200 μL Ferric alum indicator
    3. Titrate with your NH4SCN solution
    4. Your solution should turn red around 15 mL. Volume required for first titration=15.5mL. Second titration=15.5mL'
    • AgNO3+NH4SCN→AgSCN+NH4NO3
      • Reaction is 1:1
      • 2mL of AgNO3=1.5×10-4 mol
      • 15.5 mL of NH4SCN contain 1.5×10-4mol
      • [NH4SCN]=9.68 mM
  • Concentration of AgNO3 at the lowest saturated concentration:
    • [Ag][SCN]=Ksp where Ksp=1.03×10-12 (at 25ºC)
    • x2=Ksp
    • [Ag] at the lowest saturated concentration=1.01μM
  • KI Standardization
    1. Add 3 mL 1 M HNO3 and 1 mL KI to 10 mL DI H2O
    2. Add 2 mL AgNO3 and swirl to complete precipitation
    3. Add 200 μL Ferric alum indicator
    4. Titrate with your NH4SCN solution
    5. Your solution should turn red around 10 mL. Volume required for titration in first titration=10.3mL. Second titration=10.5mL
  • KI+AgNO3→AgI+AgNO3+KNO3
  • AgNO3+NH4SCN→AgSCN+NH4NO3
    • 10.4 mL of NH4SCN were required to titrate excess AgNO3. (=1.00672×10-4mol)
    • 2mL AgNO3 contain 1.5×10-4 mol
    • 1.5×10-4−1.00672×10-4=4.9328×10-5 mol of AgNO3 reacted with KI in a 1:1 reaction
    • 1 mL of KI contains 4.9328×10-5 mol
  • [KI]=49.328 mM
  • [I-] for dialysis
    • Follow the same procedure as your KI standardization
    • Note that your limit of detection will be based on how small a volume you can read (0.1 mL = 1 mM LOD)
    • You may want to try a new NH4SCN solution (more dilute) for smaller concentrations.

Fluorescence of protein solutions

  • Measured fluorescence of dyalised solutions from Oct.1
  • Fluorescence (glass microcuvette & 100 fold diluted) for all protein solutions
    • 10μL of dialysed colloid or lysozyme were diluted to 1mL by adding distilled water. (Remember to account for dilution factor later on)

Preparation of new Dialysis with 0.6 g/ L Lysozyme vs KI using 3500 MWCO

  • Add 1 mL Lysozyme to 5 wells on one side
  • Make serial dilutions of KI at 50 mM, 25 mM, 10 mM, 5 mM, and 2 mM
  • The wells were matched up in the following way. Note that 1 mL of the following were added to each well:
  1. 2 mM KI. Opposite to it Lysozyme 0.6g/L
  2. 5 mM KI. Opposite to it Lysozyme 0.6g/L
  3. 10 mM. Opposite to it Lysozyme 0.6g/L
  4. 25 mM KI. Opposite to it Lysozyme 0.6g/L
  5. 50 mM KI. Opposite to it Lysozyme 0.6g/L
  • Inserted screws to prevent evaporation
  • Place on low speed shaker for 1 week

Dialysis Data

Bradford Analysis

  • Only running bradford analysis of protein-containing solutions, that is Lys 1, 2, 3, 4 and 5, since the protein cannot diffuse through 3500 MWCO.
  • Remove 20 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, 20 μL solution + 200 μL diluted Bradford + 780 μL Tris/NaCl buffer
    • PS cuvettes, measuring 400 - 800 nm

Bradford analysis 081014.png

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:

Lysozyme-Bradford CV.png<br.> UV-Vis Absorption <br.> 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 - 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)

[I-] titration

    • AgNO3 + NH4SCN → AgSCN + NH4NO3
    • KI + AgNO3(excess) → AgI (s) + AgNO3 +KNO3
      • Initial AgNO3: 0.075 M AgNO3 x 0.002 L AgNO3 = 0.00015 mol AgNO3
      • AgNO3 (neutralized): 0.00968 M NH4SCN x 0.0104 L NH4SCN x (1 mol AgNO3 / 1 mol NH4SCN ) = 0.000100672 mol AgNO3
      • Moles of KI: 0.00015 mol AgNO3 initial - 0.000100672 mol AgNO3 neutralized = 0.0000493 mol KI
      • [KI]= 0.0000493 mol KI / 0.001 L KI = 49.3 mM KI

Titrations were performed as follows:

  • 9.68 mM NH4SCN was titrated against 3 mL 1M HNO3, 10 mL deionized H2O , 200 μLFerric alum indicator, and 0.5 mL of dialyzed sample
    • Lysozyme against 2 mM KI
      • 15.1 mL NH4SCN used
    • Lysozyme against 5 mM KI
      • 14.2 mL NH4SCN used
    • Lysozyme against 10 mM KI
      • 14.0 mL NH4SCN used
    • Lysozyme against 25 mM KI
      • 13.7 mL NH4SCN used
    • Lysozyme against 50 mM KI
      • 12.5 mL NH4SCN used
    • 2 mM KI against Lysozyme
      • 13.6 mL NH4SCN used
    • 5 mM KI against Lysozyme
      • 13.4 mL NH4SCN used
    • 10 mM KI against Lysozyme
      • 13.2 mL NH4SCN used
    • 25 mM KI against Lysozyme
      • 12.6 mL NH4SCN used
    • 50 mM KI against Lysozyme
      • 11.9 mL NH4SCN used