Product Pathways - PathScan ELISA
PathScan® Total Bad Sandwich ELISA Kit #7162
|7162S||1 Kit (96 assays)||---||In Stock||---|
|7162||carrier free and custom formulation / quantity||email request|
When ordering five or more kits, please contact us for processing time and pricing at email@example.com.
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|Kit Includes||Volume||Solution Color|
|Bad Ab-Coated Microwells||96 tests|
|Bad Detection Ab||11 ml||Green|
|Anti-mouse IgG, HRP-linked Antibody||11 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|
|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.
Storage: Kit should be stored at 4°C with the exception of Cell Lysis Buffer, which is stored at –20°C (packaged separately).
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 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. 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.
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).
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.
Have you published research involving the use of our products? If so we'd love to hear about it. Please let us know!
- 7182 PathScan® Phospho-Bad (Ser112) Sandwich ELISA Kit
- 5284 Phospho-Bad (Ser112) (40A9) Rabbit mAb
- 9296 Phospho-Bad (Ser112) (7E11) Mouse mAb
- 9291 Phospho-Bad (Ser112) Antibody
- 5286 Phospho-Bad (Ser136) (185D10) Rabbit mAb
- 4366 Phospho-Bad (Ser136) (D25H8) Rabbit mAb
- 9295 Phospho-Bad (Ser136) Antibody
- 9297 Phospho-Bad (Ser155) Antibody
- 9268 Bad (11E3) Rabbit mAb (IP Preferred)
- 9239 Bad (D24A9) Rabbit mAb
- 9292 Bad Antibody
- 9803 Cell Lysis Buffer (10X)
- 7076 Anti-mouse IgG, HRP-linked Antibody
- 7004 TMB Substrate
- 7002 STOP Solution
For Research Use Only. Not For Use In Diagnostic Procedures.
PathScan® is a trademark of Cell Signaling Technology, Inc.
Cell Signaling Technology® is a trademark of Cell Signaling Technology, Inc.