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| | ELISA Assay to Measure Human Heme Oxygenase-1 (HMOX-1) in HepG2 lysate |
| Description: | Measurement of HMOX1 ( HO-1 ) 1. In a polypropylene 384 well plate, human HO-1(after reconstitution in PBS+0.5% Triton+1 mM EDTA) was serially diluted from 20 ng/ml 2-fold in PBS+0.5% triton-X100, 1 mM EDTA to make a 12-point standard curve including a zero point. 2. In a polypropylene 96 well plate, HepG2 lysate was diluted 1 to 20 in Diluent #4 from DUOSET kit in which the assay standard is also diluted. 3. The PBS+1% BSA was emptied from the ELISA plate and 30 μl of all samples and standards were added (duplicates recommended). The plate was incubated 90 minutes. 4. Emptied plate and washed 4 times with PBST. 5. Diluted HO-1 detection antibody to 200 ng/ml in PBS+1% BSA and added 30 μl to all wells. Incubated 90 minutes. 6. Emptied plate and washed 4 times with PBST. 7. Added 30 μl streptavidin HRP at 1/200 in PBS+1% BSA (stock streptavidin concentration not specified) to all wells. Incubated 30 minutes. 8. Emptied plate and washed 4 times with PBST. 9. Diluted Lumiglo reserve reagent two parts buffer to one part lumiglo substrate. Added 30 μl to all wells. Incubated 5 minutes. 10. Measured chemiluminescence on spectramax M5 using 150 ms integration. |
| Affinity data for this assay | |
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