Product Pathways - Apoptosis
PathScan® Phospho-Bcl-2 (Ser70) Sandwich ELISA Kit #11874
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|Kit Includes||Volume||Solution Color|
|Phospho-Bcl-2(Ser70) Rabbit Ab Coated Microwells||96 tests|
|Bcl-2 Mouse 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|
|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).
Species enclosed in parentheses are predicted to react based on 100% sequence homology.
The PathScan® Phospho-Bcl-2 (Ser70) Sandwich ELISA Kit is a solid phase sandwich enzyme-linked immunosorbent assay (ELISA) that detects endogenous levels of Bcl-2 when phosphorylated at Ser70. A Phospho-Bcl-2 (Ser70) Rabbit mAb has been coated onto the microwells. After incubation with cell lysates, phosphorylated Bcl-2 protein is captured by the coated antibody. Following extensive washing, a Bcl-2 Mouse Detection mAb is added to detect the captured phospho-Bcl-2 (Ser70) 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 the developed color is proportional to the quantity of Bcl-2 phosphorylated at Ser70.Antibodies in kit are custom formulations specific to kit.
Specificity / Sensitivity
The PathScan® Phospho-Bcl-2 (Ser70) Sandwich ELISA Kit recognizes endogenous levels of Bcl-2 protein when phosphorylated at Ser70 as shown in Figure 1. Kit sensitivity is shown in Figure 2. This kit detects proteins from the indicated species, as determined through in-house testing, but may also detect homologous proteins from other species.
Figure 2. The relationship between protein concentration of lysate from Jurkat cells, untreated or treated with λ phosphatase or Paclitaxel #9807 (1 mM, 20 hr, 37ºC), and the absorbance at 450 nm is shown.
ELISA - Western correlation
Figure 1. Treatment of Jurkat cells with Paclitaxel stimulates phosphorylation of Bcl-2 at Ser70, as detected by the PathScan® Phospho-Bcl-2 (Ser70) Sandwich ELISA Kit, but does not affect the levels of total Bcl-2 detected by PathScan® Total Bcl-2 Sandwich ELISA Kit #12030. Jurkat cells were untreated or treated with λ phosphatase or Paclitaxel #9807 (1 mM, 20 hr, 37ºC). The absorbance readings at 450 nm are shown in the top figure, while the corresponding western blots using Phospho-Bcl-2 (Ser70) (5H2) Rabbit mAb #2827 (left panel) or Bcl-2 (D55G8) Rabbit mAb (Human Specific) #4223 (right panel) are shown in the bottom figure.
Bcl-2 exerts a survival function in response to a wide range of apoptotic stimuli through inhibition of mitochondrial cytochrome c release (1). It has been implicated in modulating mitochondrial calcium homeostasis and proton flux (2). Several phosphorylation sites have been identified within Bcl-2 including Thr56, Ser70, Thr74, and Ser87 (3). It has been suggested that these phosphorylation sites may be targets of the ASK1/MKK7/JNK1 pathway and that phosphorylation of Bcl-2 may be a marker for mitotic events (4,5). Mutation of Bcl-2 at Thr56 or Ser87 inhibits its anti-apoptotic activity during glucocorticoid-induced apoptosis of T lymphocytes (6). Interleukin-3 and JNK-induced Bcl-2 phosphorylation at Ser70 may be required for its enhanced anti-apoptotic functions (7).
- Murphy, K.M. et al. (2000) Cell Death Differ 7, 102-11.
- Zhu, L. et al. (1999) J Biol Chem 274, 33267-73.
- Maundrell, K. et al. (1997) J Biol Chem 272, 25238-42.
- Yamamoto, K. et al. (1999) Mol Cell Biol 19, 8469-78.
- Ling, Y.H. et al. (1998) J Biol Chem 273, 18984-91.
- Huang, S.T. and Cidlowski, J.A. (2002) FASEB J 16, 825-32.
- Deng, X. et al. (2001) J Biol Chem 276, 23681-8.
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