Product Pathways - PathScan ELISA
PathScan® Phospho-Bad (Ser112) Sandwich ELISA Kit #7182
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|Kit Includes||Volume||Solution Color|
|Bad Rabbit mAb Coated Microwells||96 tests|
|P-Bad (S112) Mouse Detection Ab||11 ml||Green|
|Anti-mouse IgG, HRP-linked Antibody||0.5 ml||Red|
|TMB Substrate #7004||11 ml||Colorless|
|STOP Solution #7002||11 ml||Colorless|
|Sealing Tape||2 sheets|
|ELISA Wash Buffer (20X)||25 ml||Colorless|
|ELISA Sample Diluent||25 ml||Blue|
|Cell Lysis Buffer (10X) #9803||15 ml||Yellowish|
Note: 12 8-well modules –Each module is designed to break apart for 8 tests.
Note: Kit should be stored at 4°C with the exception of Cell Lysis Buffer, which is stored at –20°C (packaged separately).
H M Mk
Species enclosed in parentheses are predicted to react based on 100% sequence homology.
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 #7076 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.
Specificity / Sensitivity
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.
ELISA - Western correlation
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.
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).
- Yang, E. et al. (1995) Cell 80, 285-291.
- Zha, J. et al. (1996) Cell 87, 619-628.
- Datta, S.R. et al. (1997) Cell 91, 231-241.
- Peso, L. et al. (1997) Science 278, 687-689.
- Bonni, A. et al. (1999) Science 286, 1358-1362.
- Tan, Y. et al. (1999) J. Biol. Chem. 274, 34859-34867.
- Harada, H. et al. (1999) Mol. Cell 3, 413-422.
- Tan, Y. et al. (2000) J. Biol. Chem. 275, 25865-25869.
- Lizcano, J. et al. (2000) Biochem. J. 349, 547-557.
- Datta, S. et al. (2000) Mol. Cell 6, 41-51.
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- 7162 PathScan® Total Bad Sandwich ELISA Kit
- 5284 Phospho-Bad (Ser112) (40A9) Rabbit mAb
- 9296 Phospho-Bad (Ser112) (7E11) Mouse mAb
- 9291 Phospho-Bad (Ser112) Antibody
- 9295 Phospho-Bad (Ser136) Antibody
- 9297 Phospho-Bad (Ser155) Antibody
- 9292 Bad Antibody
- 9268 Bad (11E3) Rabbit mAb (IP Preferred)
- 7076 Anti-mouse IgG, HRP-linked Antibody
- 9803 Cell Lysis Buffer (10X)
- 7004 TMB Substrate
- 7002 STOP Solution
Rabbit Monoclonals are produced under license (granting certain rights, including those under U.S. Patent No. 5,675,063) from Epitomics, Inc.
For Research Use Only. Not For Use In Diagnostic Procedures.