User:Khyra A. Neal/Notebook/Chem 571/2014/10/15

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October 15, 2014

Tasklist

Electrophoresis

  • Warm solutions for 5 minutes at 40°C that were prepared on October 14, 2014
  • Load samples to premade gel
    • Well 1: Precision Plus Protein All Blue Standards
    • Well 3: 0.12 g/L lysozyme Unk A
    • Well 4: 30:1 Lysozyme colloid
    • Well 5: 0.12 g/L Lysozyme
    • Well 6: 0.6 g/L Lysozyme
    • Wells 8-12 contain solutions prepared by Dr. Fox
  • Run for 40 minutes at 200V
  • Develop/Stain your gel
    • Place gel in Fixative Solution (40% methanol, 10% acetic acid, 50% water) for 30 minutes
    • Place gel in Stain Solution (0.025% (w/v) Coomassie Blue, 10% acetic acid, 90% water) for 1 hour
    • Place gel in Destain Solution (10% acetic acid, 90% water) for 15 minutes
      • Repeat this step with fresh destain solution 2 more times

The original protocol for electrophoresis was written by Dr. Hartings

Bradford Analysis

Bradford Analysis was conducted on the dialyzed 30:1 colloid against CaCl2 solutions

  • 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
  • Dilute to 1 mL by adding 780 μL stock 50 mM Tris/NaCl
  • Measure UV-Vis from 400 nm - 800 nm using polystyrene cuvettes.
  • Run blank of Tris/NaCl buffer
  • Run UV-Vis of undialyzed 30:1 colloid solution with Bradford reagent

Bradford of Colloid in CaCl2.jpg


NOTE The observed dialysis solution of 50 mM CaCl2 was colorless and the colloid precipitated out. Free Lysozyme is detected at 50 mM CaCl2 because the colloid precipitated out of solution.

UV-Vis and Fluorescence of Dialyzed Colloid Solutions

Fluorescence

  • Dilute Lysozyme sample 100x for each well.
  • Transfer 100 μL to a small volume fluorescence cuvette
  • Run fluorescence from 300 nm -550 nm and excitation at 280 nm
  • Clean the cuvette after each use with several SDS, HCl, HPLC, & methanol washes

UV-Vis of Colloid Solutions

  • Transfer 100 μL of dialyzed solution to small volume glass cuvette
  • Run Uv-Vis on protein containing solutions

Run from 200 nm -400 nm

Ca2+ ISE

ISE for protein containing Ca2+ ions

Ca2+ ISE

CaCl2 concentration mV
5μM 31.8
50μM 40.6
500μM 40.1
5mM 56.6
50mM 78.5

Determination of Dialyzed KI Samples extracted on October 14, 2014

Determination of KI Concentration by Titration

  • Standardization:
    • 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

NOTE The final concentration of NH4SCN was determined by titration standardization on October 8, 2014


Titrations were performed as follows

  • 9.68 mM NH4 is being 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
      • [KI] = 7.6 mM
    • Lysozyme against 5 mM KI
      • 14.2 mL NH4SCN used
      • [KI] = 25 mM
    • Lysozyme against 10 mM KI
      • 14.0 mL NH4SCN used
      • [KI] = 29 mM
    • Lysozyme against 25 mM KI
      • 13.7 mL NH4SCN used
      • [KI] = 34.8 mM
    • Lysozyme against 50 mM KI
      • 12.5 mL NH4SCN used
      • [KI] = 58 mM
    • 2 mM KI against Lysozyme
      • 13.6 mL NH4SCN used
      • [KI] = 36.7 mM
    • 5 mM KI against Lysozyme
      • 13.4 mL NH4SCN used
      • [KI] = 40.6 mM
    • 10 mM KI against Lysozyme
      • 13.2 mL NH4SCN used
      • [KI] = 44.4 mM
    • 25 mM KI against Lysozyme
      • 12.6 mL NH4SCN used
      • [KI] = 56.1 mM
    • 50 mM KI against Lysozyme
      • 11.9 mL NH4SCN used
      • [KI] = 69.6 mM

NOTE Our values are indicative of experimental error. We found that our initial dilutions of KI (2-25 mM) were not prepared at the indicated concentrations. All diluted KI solutions were discarded and new solutions were prepared using fresh 50 mM KI on October 21, 2014.