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Sandwich ELISA analysis of Human GFAP matched pair antibodies
Elisa assay was performed by coating wells of a 96-well plate with 100 µl per well of capture antibody (HA722234) 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 human GFAP protein (HA210990) starting from 100000 pg/ml to 0 pg/ml and detect antibody (HA601342, 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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The concentrations of GFAP were interpolated from the GFAP standard curve and corrected for sample dilution. The mean GFAP concentration was determined to be 41.29 ng/ml in rat brain tissue extract. There was no detectable signal in 293T and NIH/3T3 cell extract.
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