Figure 1. Treatment of OVCAR8 cells with TPA stimulates phosphorylation of Bad at Ser112, detected by PathScan ® Phospho-Bad (Ser112) Sandwich ELISA Kit #7182, but does not affect the level of total Bad protein detected by PathScan ® Total Bad Sandwich ELISA Kit #7162. Lambda phosphatase treatment of control cell lysates (4000 U/mL for 60 minutes at 37ºC) abolishes the basal phosphorylation of Bad as shown by both Sandwich ELISA and Western analysis. The absorbance readings at 450 nm are shown in the top figure, while the corresponding Western blots using Phospho-Bad (Ser112) Antibody #9296 (right panel) or Bad Antibody #9254 (left panel), are shown in the bottom figure.
Figure 2. The relationship between the protein concentration of untreated and TPA-treated OVCAR8 cell lysates and the absorbance at 450 nm is shown. Cells were serum starved overnight and then treated with 200 nm TPA for 30 min. at 37ºC.
Figure 3. Kit specificity as demonstrated by Western analysis of the ELISA microwell captured protein. Lysates were prepared from OVCAR8 cells and incubated in microwells coated with the Bad capture antibody. Wells were washed, and the captured protein was solubilized in SDS gel loading buffer. Western analysis of OVCAR8 cell starting lysate (lanes 1 & 2) and the captured protein (lanes 3 & 4) was performed using Bad Mouse mAb #9254. The major band detected in the captured material corresponds to the Bad protein (lanes 3 & 4).