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 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 1-4 column 5-8 column 9-12
0 24hrs PBS 24hrs IFNg
10 48hr PBS 48hrs IFNg
25 72hrs PBS 72hrs IFNg
50
75 24hrs LPS 24hrs LPS+IFNg
100 48hrs LPS 48hrs LPS+IFNg
125 72hrs LPS 72hrs 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)
0 0 1000
10 34.5 965.5
25 8602 913.8
50 172.5 827.5
75 258.6 741.4
100 345 655
125 431 569
250 862 138
  • 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
A 0.069 0.066 0.071 0.066 0.062 0.068 0.183 0.105 0.064 0.422 0.062 0.519
B 0.076 0.079 0.078 0.079 0.059 0.058 0.424 0.047 0.332 0.09 0.124 0.485
C 0.118 0.113 0.122 0.115 0.061 0.054 0.049 0.044 0.123 0.323 0.595 0.447
D 0.151 0.167 0.18 0.161 0.036 0.049 0.034 0.032 0.03 0.029 0.029 0.032
E 0.208 0.201 0.259 0.254 0.094 0.086 0.093 0.086 0.093 0.085 0.098 0.519
F 0.231 0.253 0.316 0.288 0.137 0.127 0.116 0.29 0.117 0.125 0.111 0.377
G 0.264 0.264 0.341 0.282 0.111 0.107 0.084 0.161 0.145 0.179 0.135 0.168
H 0.383 0.33 0.33 0.295 0.033 0.028 0.024 0.024 0.024 0.026 0.029 0.027


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