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 NaNO2 24 hr (-) control (PBS) 24 hr (+) LPS
10 µM NaNO2 48 hr (-) control (PBS) 48 hr (+) LPS
25 µM NaNO2 72 hr (-) control (PBS) 72 hr (+) LPS
50 µM NaNO2 Nothing Nothing
75 µM NaNO2 24 hr (+) IFN-g 24 hr (+) LPS (+) IFN-g
100 µM NaNO2 48 hr (+) IFN-g 48 hr (+) LPS (+) IFN-g
125 µM NaNO2 72 hr (+) IFN-g 72 hr (+) LPS (+) IFN-g
250 µM NaNO2 Nothing Nothing

Calculations for NaNO2

Raw Data

Row\Column 1 2 3 4 5 6 7 8 9 10 11 12
A 0.062 0.057 0.056 0.051 0.044 0.042 0.037 0.037 0.079 0.072 0.076 0.063
B 0.063 0.061 0.059 0.052 0.042 0.04 0.036 0.034 0.116 0.12 0.118 0.124
C 0.077 0.077 0.079 0.076 0.042 0.04 0.041 0.036 0.15 0.144 0.138 0.16
D 0.135 0.119 0.123 0.114 0.026 0.026 0.026 0.03 0.034 0.025 0.029 0.044
E 0.174 0.174 0.173 0.152 0.063 0.052 0.051 0.054 0.057 0.077 0.07 0.079
F 0.188 0.197 0.178 0.179 0.093 0.09 0.09 0.089 0.157 0.137 0.086 0.112
G 0.189 0.226 0.186 0.194 0.105 0.103 0.128 0.131 0.165 0.106 0.092 0.11
H 0.189 0.294 0.299 0.303 0.036 0.048 0.052 0.051 0.036 0.074 0.064 0.101

Standard Curve Values and Calculations

Standard Curve Concentration of NaNO2 (µM) Average Absorbance (A595) 100X Average Absorbance (A595)£
0 µM NaNO2 0 0.0565 5.65
10 µM NaNO2 10 0.05875 5.875
25 µM NaNO2 25 0.07725 7.725
50 µM NaNO2 50 0.12275 12.275
75 µM NaNO2 75 0.16825 16.825
100 µM NaNO2 100 0.1855 18.55
125 µM NaNO2 125 0.19875 19.875
250 µM NaNO2 250 0.27125 27.125
  • £ Used to calculate standard curve more accurately

Standard Curve

090331 NO Assay Standard Curve.png

Sample Concentrations

Sample Average Absorbance (A595) 100X Average Abs (A595)** Calculated Concentration (µM) Normalized Concentrations (µM)*
24 hr (-) control (PBS) 0.04 4 -33.96 0.00
48 hr (-) control (PBS) 0.038 3.8 -36.18 0.00
72 hr (-) control (PBS) 0.03975 3.975 -34.24 0.00
24 hr (+) IFN-g 0.055 5.5 -17.35 0.00
48 hr (+) IFN-g 0.0905 9.05 21.96 21.96
72 hr (+) IFN-g 0.11675 11.675 51.03 51.03
24 hr (+) LPS 0.0725 7.25 2.03 2.03
48 hr (+) LPS 0.1195 11.95 54.08 54.08
72 hr (+) LPS 0.148 14.8 85.64 85.64
24 hr (+) LPS (+) IFN-g 0.07075 7.075 0.09 0.09
48 hr (+) LPS (+) IFN-g 0.123 12.3 57.95 57.95
72 hr (+) LPS (+) IFN-g 0.11825 11.825 52.69 52.69
  • *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

090331 NO Concentrations Timecourse.png

Calculations File

Calculations can be found in this document.