Great ELISA Kit for Measuring IGF1R Expression in Cells

Protocol Overview

The IGF-1R human ELISA kit (Abcam, Cambridge, Massachusetts, USA) was used to quantify the amount of human IGF-1R in three pancreatic cancer cell lines. The ELISA was performed according to the manufacture’s protocol. Briefly, cells were first lysed with RIPA buffer and total protein was measured as described for the Western blot. IGF-1R standards were made from 0 to 6000 pg/mL. Next, 100 µL of standards and samples were added into appropriate wells. The plate was covered and incubated for 12 h at 4 ºC with gentle shaking. The solution was discarded and wells were washed four times with 1X Wash Solution. Next, 100 µL of 1X Biotinylated IGF1R Detection Antibody was added to each well. The plate was incubated for 1 h with gentle shaking at room temperature. The solution was removed and the plate was washed four times with 1X Wash Solution. Next, 100 µL of 1X HRP-Streptavidin solution was added to each well and the plate was incubated for 45 min at room temperature. The solution was discarded and the plate was washed four times with 1X Wash Solution. Next, 100 µL of TMP One Step Substrate Reagent was added to each well and the plate was incubated in the dark for 30 min at room temperature. Lastly, 50 µL of Stop Solution was added to the corresponding wells and measurements were taken at 450 nm.