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 lysates from untreated and TPA-treated OVCAR8 cells 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.
|REACTIVITY||H M Mk|
|Product Includes||Volume||Solution Color|
|Bad Rabbit mAb Coated Microwells||96 tests|
|Phospho-Bad (Ser112) Mouse Detection mAb||1 ea||Green (Lyophilized)|
|Anti-mouse IgG, HRP-linked Antibody (ELISA Formulated)||1 ea||Red (Lyophilized)|
|Detection Antibody Diluent||11 ml||Green|
|HRP Diluent||11 ml||Red|
|TMB Substrate 7004||11 ml|
|STOP Solution 7002||11 ml|
|Sealing Tape||2 ea|
|ELISA Wash Buffer (20X) 9801||25 ml|
|ELISA Sample Diluent||25 ml||Blue|
|Cell Lysis Buffer (10X) 9803||15 ml|
CST's PathScan® Phospho-Bad (Ser112) Sandwich ELISA Kit is a solid phase sandwich enzyme-linked immunosorbent assay (ELISA) that detects endogenous levels of phospho-Bad (Ser112) protein. A Bad rabbit mAb has been coated onto the microwells. After incubation with cell lysates, Bad protein (phospho and nonphospho) is captured by the coated antibody. Following extensive washing, a phospho-Bad (Ser112) mouse mAb is added to detect the captured phospho-Bad protein. Anti-mouse IgG, HRP-linked Antibody is then used to recognize the bound detection antibody. HRP substrate, TMB, is added to develop color. The magnitude of absorbance for this developed color is proportional to the quantity of phospho-Bad (Ser112) protein.
Antibodies in kit are custom formulations specific to kit.
NOTE: Prepare solutions with purified water.
*NOTE: Some PathScan® ELISA Kits may include HRP-Linked Streptavidin in place of HRP-Linked Antibody.
NOTE: Initial color of positive reaction is blue, which changes to yellow upon addition of STOP Solution.
posted November 2013
Protocol Id: 204
CST's PathScan® Phospho-Bad (Ser112) Sandwich ELISA Kit #7182 detects endogenous levels of phospho-Bad (Ser112) protein. A significant induction of Bad phosphorylation at Ser112 can be detected in TPA-treated OVCAR8 cells using PathScan® Phospho-Bad (Ser112) Sandwich ELISA Kit #7182. However, the level of total Bad protein (phospho and nonphospho) detected by PathScan® Total Bad Sandwich ELISA Kit #7162 remains unchanged (Figure 1). This kit detects proteins from the indicated species, as determined through in-house testing, but may also detect homologous proteins from other species.
Human, Mouse, Monkey
Bad is a proapoptotic member of the Bcl-2 family that promotes cell death by displacing Bax from binding to Bcl-2 and Bcl-xL (1,2). Survival factors, such as IL-3, inhibit the apoptotic activity of Bad by activating intracellular signaling pathways that result in the phosphorylation of Bad at Ser112 and Ser136 (2). Phosphorylation at these sites promotes binding of Bad to 14-3-3 proteins to prevent an association between Bad with Bcl-2 and Bcl-xL (2). Akt phosphorylates Bad at Ser136 to promote cell survival (3,4). Bad is phosphorylated at Ser112 both in vivo and in vitro by p90RSK (5,6) and mitochondria-anchored PKA (7). Phosphorylation at Ser155 in the BH3 domain by PKA plays a critical role in blocking the dimerization of Bad and Bcl-xL (8-10).
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