User:Jacob Esenther/Notebook/Chem 571/2014/11/12

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Objective

  • Run all tests on the Lysozyme-CaCl2 dialysis
  • Make more lysozyme colloid solution
  • Set up a dialysis of Lysozyme-CaCl2 with new concentrations

Description

  1. Colloid Synthesis
    1. make 0.04621g HAuCl4 / 50mL H2O
    2. make 0.0448g Lysozyme / 25mL H2O
    3. make a 30:1 HAuCl4 Lysozyme solution
      • 0.5mL HAuCl4, 4.17mL Lysozyme, and 0.33mL 50mL H2O
    4. Heat in an oven over 4 hours at 80°C
  2. Its go time - 0.6g Lysozyme against 50mM, 5mM, 500μM, 50μM, 5μM CaCl2
    1. Bradford Analysis
      • 50μL sample
      • 200μL Bradford reagent
        • 4:1 DI Water to Bradford Assay
      • 750μL Tris-NaCl Buffer
        • 50mM Tris + 50mM NaCl
    2. UV Protein Analysis
      • 10:1 dilutions
    3. Fluorescence Analysis
      • 10:1 dilutions
    4. ISC of CaCl2
      • The probe was allowed to sit in solution before taking measurements, this seemed to yield better results. These measurements may need to be taken again to double check

Data

  1. Bradford Analysis

Graph plotting Absorbance of Lysozyme at differing Wavelengths

' ' y=0.0206x+0.0969
Abs. at 590nM Concentration of Lysozyme (ug/mL)
Bradford Blank 0.204 5.199029126
5uM CaCl2 0.46 17.62621359
50uM CaCl2 0.467 17.96601942
500uM CaCl2 0.431 16.2184466
5mM CaCl2 0.466 17.91747573
50mM CaCl2 0.43 16.16990291
  • Table Displaying Calculated Concentrations based off of a Calibration curve of absorbance against a known concentration of Lysozyme.
  1. UV Protein Analysis

Plot of absorbance of Lysozyme in the UV range of 200-400nm

  1. Fluorescence Analysis

Graph plotting Fluorescence at different Wavelength

  1. ISC of CaCl2
' ISC - CaCl2 Potential=26.344(Concentration)+40.946 ' ' '
Theoretical Concentration (mM) Potential (mV) log (concentration) Actual Concentration (mM)
Samples 50.000 89.7 1.850668084 70.90356695
5.000 65.6 0.93584877 8.62678094
0.500 46 0.191846341 1.555415207
0.050 32.1 -0.335788035 0.461542783
0.005 26.1 -0.563543881 0.273184541
Stocks 50.000 93.8 2.006301245 101.4614922
5.000 65.9 0.947236562 8.855978681
0.500 43.8 0.108335864 1.283322663
0.050 28.5 -0.472441543 0.336944566
0.005 15 -0.984892196 0.103539915

Notes

Always discuss methods between groups. It can only help you perfect your procedures.