User:Eric Ma/Notebook/MICB323 Lab Book/2009/03/31

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Goals

  1. Perform ECL (Enhanced Chemiluminescence) Detection of iNOS/NOS from the Western Transfer blot.
  2. Perform a Nitric Oxide Assay on the LPS +/- IFNg stimulated culture supernatants

Part 1: ECL Detection of iNOS/NOS from the Western Transfer Blot

  • Procedure followed as in lab manual pg 130.

Part 2: NO Assay on the LPS+/-IFNg stimulated culture supernatants

Plate Layout

Column 1-4 Column 5-8 Column 9-12
0 µM NaNO224 hr (-) control (PBS)24 hr (+) LPS
10 µM NaNO248 hr (-) control (PBS)48 hr (+) LPS
25 µM NaNO272 hr (-) control (PBS)72 hr (+) LPS
50 µM NaNO2NothingNothing
75 µM NaNO224 hr (+) IFN-g24 hr (+) LPS (+) IFN-g
100 µM NaNO248 hr (+) IFN-g48 hr (+) LPS (+) IFN-g
125 µM NaNO272 hr (+) IFN-g72 hr (+) LPS (+) IFN-g
250 µM NaNO2NothingNothing

Calculations for NaNO2

Raw Data

Row\Column 1 2 3 4 5 6 7 8 9 10 11 12
A0.0620.0570.0560.0510.0440.0420.0370.0370.0790.0720.0760.063
B0.0630.0610.0590.0520.0420.040.0360.0340.1160.120.1180.124
C0.0770.0770.0790.0760.0420.040.0410.0360.150.1440.1380.16
D0.1350.1190.1230.1140.0260.0260.0260.030.0340.0250.0290.044
E0.1740.1740.1730.1520.0630.0520.0510.0540.0570.0770.070.079
F0.1880.1970.1780.1790.0930.090.090.0890.1570.1370.0860.112
G0.1890.2260.1860.1940.1050.1030.1280.1310.1650.1060.0920.11
H0.1890.2940.2990.3030.0360.0480.0520.0510.0360.0740.0640.101

Standard Curve Values and Calculations

Standard Curve Concentration of NaNO2 (µM) Average Absorbance (A595) 100X Average Absorbance (A595)£
0 µM NaNO200.05655.65
10 µM NaNO2100.058755.875
25 µM NaNO2250.077257.725
50 µM NaNO2500.1227512.275
75 µM NaNO2750.1682516.825
100 µM NaNO21000.185518.55
125 µM NaNO21250.1987519.875
250 µM NaNO22500.2712527.125
  • £ Used to calculate standard curve more accurately

Standard Curve

Sample Concentrations

Sample Average Absorbance (A595) 100X Average Abs (A595)** Calculated Concentration (µM) Normalized Concentrations (µM)*
24 hr (-) control (PBS)0.044-33.960.00
48 hr (-) control (PBS)0.0383.8-36.180.00
72 hr (-) control (PBS)0.039753.975-34.240.00
24 hr (+) IFN-g0.0555.5-17.350.00
48 hr (+) IFN-g0.09059.0521.9621.96
72 hr (+) IFN-g0.1167511.67551.0351.03
24 hr (+) LPS0.07257.252.032.03
48 hr (+) LPS0.119511.9554.0854.08
72 hr (+) LPS0.14814.885.6485.64
24 hr (+) LPS (+) IFN-g0.070757.0750.090.09
48 hr (+) LPS (+) IFN-g0.12312.357.9557.95
72 hr (+) LPS (+) IFN-g0.1182511.82552.6952.69
  • *Note: Normalized to "0" because it doesn't make sense to have negative concentrations.
  • **Note: 100X absorbance was used to obtain a more accurate equation for the standard curve.

Timecourse Graph of NO Production

Calculations File

Calculations can be found in this document.


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