Cell Signaling Technology

Product Pathways - PathScan ELISA

PathScan® Total Bad Sandwich ELISA Kit #7162

Kit Includes Volume Solution Color
Bad Antibody-Coated Microwells 96 tests
Bad Detection Ab 11 milliliters Green
Anti-Mouse IgG HRP-Linked Ab 11 milliliters Red
TMB Substrate 11 milliliters Colorless
STOP Solution 11 milliliters Colorless
Sealing Tape 2 sheets
20X Wash Buffer 25 milliliters Colorless
Sample Diluent 25 milliliters Blue
Cell Lysis Buffer (10X) # 9803 15 milliliters 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 (10X), which is stored at –20°C (packaged separately).

Species Cross-Reactivity

H Mk

Reactivity Key:  H=Human  Mk=Monkey

Description

CST's PathScan® Total Bad Sandwich ELISA Kit is a solid phase sandwich enzyme-linked immunosorbent assay (ELISA) that detects endogenous levels of total Bad 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 Bad mouse mAb* is added to detect the captured 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 total Bad protein.* Antibodies in kit are custom formulations specific to kit.

Specificity / Sensitivity

CST's PathScan® Total Bad Sandwich ELISA Kit #7162 detects endogenous levels of Bad 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). In Figure 3, Western analysis of protein captured in microwells coated with the Bad antibody shows a major band corresponding to the Bad protein.

Sandwich ELISA

Sandwich ELISA

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 37°C) 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.

Sandwich ELISA

Sandwich ELISA

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 37°C.

Sandwich ELISA

Sandwich ELISA

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).


Background

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 protein to prevent an association between Bad and 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 of Ser155 in the BH3 domain by PKA plays a critical role in blocking the dimerization of Bad and Bcl-xL (8-10).

  1. Yang, E. et al. (1995) Cell 80, 285-291.
  2. Zha, J. et al. (1996) Cell 87, 619-628.
  3. Datta, S.R. et al. (1997) Cell 91, 231-241.
  4. Peso, L. et al. (1997) Science 278, 687-689.
  5. Bonni, A. et al. (1999) Science 286, 1358-1362.
  6. Tan, Y. et al. (1999) J. Biol. Chem. 274, 34859-34867.
  7. Harada, H. et al. (1999) Mol. Cell 3, 413-422.
  8. Tan, Y. et al. (2000) J. Biol. Chem. 275, 25865-25869.
  9. Lizcano, J. et al. (2000) Biochem. J. 349, 547-557.
  10. Datta, S. et al. (2000) Mol. Cell 6, 41-51.

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