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Sandwich ELISA analysis of Mouse IL-12p40 matched pair antibodies
Elisa assay was performed by coating wells of a 96-well plate with 100 µl per well of capture antibody (HA722193) diluted in carbonate/bicarbonate buffer, at a concentration of 5 µg/ml overnight at 4℃. Wells of the plate were washed, blocked with 150 µl 0.05% tween-20 1%BSA blocking buffer, and incubated with serial diluted Recombinant mouse IL-12p40 protein (HA210890) starting from 3000 pg/ml to 0 pg/ml and detect antibody (HA722194, Biotin, 0.2 µg/ml) for 1 hour at 30℃ with shaking. Then the plate was washed and incubated with 100 µl per well of SA-HRP for 0.5 hour at 30℃ with shaking. Detection was performed using an Ultra TMB Substrate for 10 minutes at room temperature in the dark. The reaction was stopped with sulfuric acid and absorbances were read on a spectrophotometer at 450 nm.
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Interpolated concentrations of native IL-12 p40 in cell culture supernatant and mouse serum samples.
IL-12 p40 was measured in 100% cell supernatant. The concentrations of IL-12 p40 were interpolated from the IL-12 p40 standard curves. The mean IL-12 p40 concentration was determined to be 4055 pg/mL in mouse serum. There was no detectable signal in NIH/3T3 cell supernatant.
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Interpolated concentrations of spiked IL-12 p40 in mouse cell culture media samples.
The concentrations of IL-12 p40 were measured in duplicates, interpolated from the IL-12 p40 standard curves and corrected for sample dilution. Undiluted samples are as follows: cell culture media 25%.
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Interpolated concentrations of native IL-12 p40 in mouse serum sample.
The concentrations of IL-12 p40 were interpolated from the IL-12 p40 standard curves and corrected for sample dilution. Undiluted samples are serum 100%. The mean IL-12 p40 concentration was determined to be 4055 pg/mL in mouse serum.
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