User:Monika Gasiorek/Notebook/CHEM-571 2014F/2014/11/11

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November 11, 2014

Bradford Calibration Curve - Take 2

Due to poor results in the past, Paul made a new Bradford calibration curve using varying amounts of 0.12 g/L lysozyme stock solution. He used varying amounts of the stock combined with 200 µL of Bradford reagent and enough 50 mM TRIS/50 mM NaCl buffer for a total sample volume of 1 mL.

Volume of Stock Final Concentration
20 µL 2.4 µg/mL
40 µL 4.8 µg/mL
60 µL 7.2 µg/mL
80 µL 9.6 µg/mL
100 µL 12 µg/mL

Results

11.11.14 Bradford Calibration Spectra MGPR.png

11.11.14 Bradford Calibration Curve MGPR.png

CaCl2 ISE Calibration Curve - Take 2

For the same reason stated above, Paul also made a new Ca2+ by determining the potentials of the original 0.005, 0.05, 0.5, 5, and 50 mM CaCl2 solutions in addition to the 5, 15, 25, 35, and 45 mM CaCl2 solutions made last week.

Concentration (mM) Potential (mV)
0.005 (-17.4)
0.05 3.3
0.5 33.5
5 59.6
15 71.8
25 78.7
35 82.7
45 85.2
50 84.1


CaCl2 Calibration Curve.png

Primary Experiment #3 - Take 2

0.6 g/L Lysozyme vs CaCl2 using MWCO 3500

Well 1 Well 2 Well 3 Well 4 Well 5
0.6 g/L lysozyme 0.6 g/L lysozyme 0.6 g/L lysozyme 0.6 g/L lysozyme 0.6 g/L lysozome
45 mM CaCl2 35 mM CaCl2 25 mM CaCl2 15 mM CaCl2 5 mM CaCl2

Bradford Analysis

30 µL samples of each of the dialyzed protein-containing samples were combined with 200 µL of Bradford reagent and 770 µL of 50 mM TRIS/50 mM NaCl buffer in order to run absorbance spectra, demonstrated numerically and graphically below:

Bradford CaCl2-Lys Spectra 11.11.14.png

Bradford Values.png

Fluorescence

10 µL samples of each of the dialyzed protein-containing samples were diluted with 990 µL of water in order to obtain fluorescence emission spectra at an excitation wavelength of 280 nm.

Fluorescence CaCl2-Lys Spectra 11.11.14.png

Ca2+ ISE

The following values correlate to starting CaCl2 solution concentrations of 5, 15, 25, 35, and 45 mM, respectively, top to bottom.

Ca ISE values.png

Primary Experiment #2

I set up another dialysis for 30:1 colloid vs. CaCl2 - 3500 MWCO

Well 1 Well 2 Well 3 Well 4 Well 5
30:1 colloid 30:1 colloid 30:1 colloid 30:1 colloid 30:1 colloid
45 mM CaCl2 35 mM CaCl2 25 mM CaCl2 15 mM CaCl2 5 mM CaCl2

NOTE: I ran out of colloid; the well across from 45 mM CaCl2 was about half empty.