ECL of iNOS and Nitric Oxide Assay
ECL
1o incubation: 1:09->2:07
3 washes
2o incubation:2:20
- 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
|
- 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
|
Project name
