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.

  • The Bradford reagant was prepared by a 1:4 dilution with previously made 50 mM Tris/50 mM NaCl solution

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:

  • Two additional samples were run, including the 0.5 g/L lysozyme stock solution and a bradford reagent & Tris/NaCl buffer blank

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:

Wavelength 575 Calculated Concentrations (g/L)
Stock0.1050.608040201
50 mM CaCl20.1080.75879397
50 mM CaCl2 Soak0.012-4.065326633
500 μM CaCl20.1030.507537688
500 μM CaCl2 Soak-0.009-5.120603015
HCl0.092-0.045226131
HCl Soak-0.005-4.91959799
Water0.1120.959798995
Water Soak-0.005-4.91959799

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.

  • The following table demonstrates the organization/content of each side of the five well chamber:

Well 1 Well 2 Well 3 Well 4 Well 5
0.12 g/L lysozyme 0.12 g/L lysozyme 0.12 g.L lysozyme 30:1 [Au]:[Lys] colloid 30:1 [Au]:[Lys] colloid
50 μM CaCl2 50 mM CaCl2 500 μM CaCl2 50 μM CaCl2 50 mM CaCl2

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.

  • 1.2 mL of the stock solution was diluted with 3.8 mL of HPLC water.

NaCl Conductivity Calibration Curve

I)20 mL of a 50 mM NaCl solution was prepared.

  • 0.05844 g of NaCl salt was dissolved in 50 mL of water

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

  • 2 mL of each preceding solution was diluted with 18 mL of water

Concentration Conductivity (mV)
5 µM 179.8
50 µM 148.1
500 µM 87.5
5 mM 30.0
50 mM (-19.6)

NOTE:The results were graphed with conductivity as a function of the log of CaCl2 concentration.

Image:Cl-_Conductivity_Calibration_Curve.png

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.

  • The amount of foreign salt added to each sample was calculated in order to give a 50 mM concentration of that salt in the 50 mM NaCl solution to which it was being added

Salt Amount added (g) Conductivity (mV)
KNO3 0.10111 (-22.0)
Na2SO4 0.14204
KBr 0.11902

IV) Conductivity measurements using the Cl- probe were also obtained for 4 other standard samples, as follows:

Sample Conductivity (mV)
HPLC Water 133.1
DI Water 158.3
Tap Water 69.1
0.5 g/L Lysozyme stock solution 89.5

CaCl2 Conductivity, Continued

Conductivity measurements using the Ca2+ ISE probe were obtained for 4 other standard samples, as follows:

Sample Conductivity (mV)
HPLC Water (-78.5)
DI Water (-110.3)
Tap Water 45.7
0.5 g/L Lysozyme stock solution 85.4


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