User:Monika Gasiorek/Notebook/CHEM-571 2014F/2014/10/07

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October 7, 2014

Continuation of 3500 MWCO Dialysis Analysis

Bradford analysis of all protein-containing samples was completed last week.

Fluorescence analysis was done on all protein containing solutions of this dialysis, including:

  • 0.5 g/L lysozyme against HPLC water, 0.25 mM HCl, and 50 mM & 500 μM CaCl2

Emission spectra were obtained on a range of 300 to 550 nm with an excitation wavelength of 280 nm. The cuvette was cleaned with 1 N HCl, HPLC water, and methanol between runs to prevent protein residue build-up. Each sample was diluted 100 fold (10 μL in 990 μL).

NOTE:The same procedure was followed first for the listed soak solution samples, only realizing after obtaining each spectra that fluorescence data was only necessary for protein-containing samples.

The fluorimetry results will be posted as soon as possible.

UV Absorption spectra were obtained for each of the crude, protein-containing samples mentioned above on a 200-800 nm range. The cuvettes were cleaned in the same manner as with fluorescence.

3500MW Dialysis 1in100 NoBradford.png

Ca2+ concentration in Ca2+ containing samples (wells 4 and 5) will be calculated using our pre-determined calibration curve given the following ISE hardwater probe conductivities(?):

Concentration mV
500 µM CaCl2 34.3 mV
L-500 µM CaCl2 31.1 mV
50 mM CaCl2 77.5 mV
L-50 mM CaCl2 77.0 mV

pH measurements were obtained for all samples containing H+ ions (well 2) with the following results:

  1. HCl-Lysozyme - 7.926
  2. HCl Soak - 8.138

Colloid Synthesis

20 mL of 30:1 [Au]:[Lysozyme] of new colloid solution was made.

  • 0.5 mL of 2.512 x 10-3 HAuCl4 was combined with 1 mL of 0.6 g/L (or 42 μM) lysozyme stock solution and 3.5 mL of DI water in 4 different test tubes for a total of 20 mL
  • These test tubes were heated at 80C for 4 hours