User:Mark X. Ling/Notebook/Micb 323/2009/03/31

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ECL of iNOS and Nitric Oxide Assay

ECL

1o incubation: 1:09->2:07 3 washes 2o incubation:2:20 Image:Img108.jpg

  • as expected no bands showed up on Crystal's xray. No iNOS on PBS, and there is too little iNOS in the total protein from the lysate to be detected.
  • only one lane of LPS induced had iNOS detected.
    • used 10ng/mL only, so rest may not have been too induced
  • may have been problem with the immunoprecipitation, not enough iNOS concentrated for the rest.
  • if any was to have a band, LPS is expected => had most NO in the NO assay

Nitric Oxide Assay

  • incubated at: 1:56 -> 2:09

Multititer plate map

NaNO2 standard (uM) tissue culture samples tissue culture samples
column 1-4column 5-8column 9-12
024hrs PBS24hrs IFNg
1048hr PBS48hrs IFNg
2572hrs PBS72hrs IFNg
50
7524hrs LPS24hrs LPS+IFNg
10048hrs LPS48hrs LPS+IFNg
12572hrs LPS72hrs LPS+IFNg
250
  • note the change of the sample placement, LPS samples and IFNg treatments switched places
  • there are no 96hr samples

NaNO2 Standard

[NaNO2] (uM) amount of NaNO2 Standard (uL) Amount of dH2O (uL)
001000
1034.5965.5
258602913.8
50172.5827.5
75258.6741.4
100345655
125431569
250862138
  • 2mg tablet dissolved in 1mL water ==> solution is then diluted 1/100
    • stock concentration = 289.9uM

My results for Nitric Oxide Assay

' 1 2 3 4 5 6 7 8 9 10 11 12
A0.0690.0660.0710.0660.0620.0680.1830.1050.0640.4220.0620.519
B0.0760.0790.0780.0790.0590.0580.4240.0470.3320.090.1240.485
C0.1180.1130.1220.1150.0610.0540.0490.0440.1230.3230.5950.447
D0.1510.1670.180.1610.0360.0490.0340.0320.030.0290.0290.032
E0.2080.2010.2590.2540.0940.0860.0930.0860.0930.0850.0980.519
F0.2310.2530.3160.2880.1370.1270.1160.290.1170.1250.1110.377
G0.2640.2640.3410.2820.1110.1070.0840.1610.1450.1790.1350.168
H0.3830.330.330.2950.0330.0280.0240.0240.0240.0260.0290.027


Post lab comments

  • NO assay result were not good
    • too much bubble, so there were spots with very high reading
    • no reliable enough so used Crystal's data
  • xray result is okay, at least saw a band.
  • indicates ECL prcedure went okay.
    • immunoprecipitation mistakes, maybe only the 48 hours LPS went well


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