User:Monika Gasiorek/Notebook/CHEM-571 2014F/2014/10/01
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October 1, 2014
Bradford Analysis of Multi-Well Dialysis
I) 20 μL of each sample from each side of all four filled wells was extracted from the chamber (8 samples total).
II) 200 μL of Bradford reagent was added to each 20 μL sample.
III) 780 μL of the 50 mM Tris/500 mM NaCl solution was also added to all 8 samples.
IV) A UV Vis spectra was obtained for each sample:
NOTE:The spectra of the soak solutions were not included because the MW cutoff of the dialysis membrane prevented the lysozyme from moving across the membrane into the soak solution and thus the absorbance/protein concentration in these samples was near to 0.
V) The previously constructed Bradford calibration curve was utilized to determine the protein concentrations in the four lysozyme-containing cells:
VI) All eight samples were extracted from the multi-well chamber and stored for further analysis to be completed next week.
Week-Long Dialysis Set-up
A second five well chamber dialysis was set up to run for the week.
I) A piece of 20K MWCO flat dialysis tubing was cut, wet, and inserted in between the two plates of a five well dialysis chamber. The dialysis chamber was then clamped shut.
II) Each side of each well of the chamber was filled with 1 mL of an appropriate sample (10 samples total) using a glass Pasteur pipette and by tilting the chamber to an angle which reduces surface tension and overflow.
III) Top screws were inserted in the openings above each side of all five wells, preventing leakage or evaporation of sample.
The CaCl2 solutions (50 mM, 50 μM, and 5μM) were prepared last week for the determination of a CaCl2 calibration curve.
Additionally, the 0.12 g/L lysozyme samples were prepared from the 0.5 g/L lysozyme stock used in the previous day's dialysis.
NaCl Conductivity Calibration Curve
I)20 mL of a 50 mM NaCl solution was prepared.
II)Four serial dilutions were then done in order to prepare 20 mL of 5 mM, 500 μM, 50 μM, and 5 μM NaCl solutions
NOTE:The results were graphed with conductivity as a function of the log of CaCl2 concentration.
III) Additional conductivity measurements were done on three separate 1-2 mL samples of the 50 mM NaCl solution to test for (ion pairing &) interference in chloride ion conductivity measurements given the presence of a non-chloride containing salt.
IV) Conductivity measurements using the Cl- probe were also obtained for 4 other standard samples, as follows:
CaCl2 Conductivity, Continued
Conductivity measurements using the Ca2+ ISE probe were obtained for 4 other standard samples, as follows: