Phospho-ERK1 (T202+Y204)/ERK2 (T185+Y187) Recombinant Rabbit Monoclonal Antibody [SC58-01] - BSA and Azide free (Capture)

Sandwich ELISA analysis of Human ERK1 (pT202+pY204)/ERK2 (pT185+pY187) protein matched pair antibodies

Capture: HA724764, Phospho-ERK1 (T202+Y204)/ERK2 (T185+Y187) Rabbit mAb [SC58-01]
Detector: HA724765, ERK1/2 Rabbit mAb [SA43-03]

Elisa assay was performed by coating wells of a 96-well plate with 100 µl per well of capture antibody (HA724764) 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 ERK1 (pT202+pY204)/ERK2 (pT185+pY187) protein (HA211528) starting from 200,000 pg/mL to 0 pg/mL and detect antibody (HA724765, 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.

Interpolated concentrations of native ERK1 (pT202+pY204)/ERK2 (pT185+pY187) in NIH/3T3 stimulated or unstimulated with 200nM PMA for 30 minutes, NIH/3T3 cell extract samples based on a 1,000 µg/mL extract load.

Capture: HA724764, Human Phospho-ERK1 (T202+Y204)/ERK2 (T185+Y187) Rabbit mAb [SC58-01]
Detector: HA724765, ERK1/2 Rabbit mAb [SA43-03]

The concentrations of ERK1 (pT202+pY204)/ERK2 (pT185+pY187) were measured in duplicates, interpolated from the ERK1 (pT202+pY204)/ERK2 (pT185+pY187) standard curve and corrected for sample dilution. The interpolated dilution factor corrected values are plotted (mean +/- SD, n=2). The mean ERK1 (pT202+pY204)/ERK2 (pT185+pY187) concentration was determined to be 116 ng/mL in stimulated NIH/3T3 cell extract and 12 ng/mL in unstimulated NIH/3T3 cell extract.