Cell Signaling Technology

Product Pathways - Apoptosis

PathScan® Total Bcl-2 Sandwich ELISA Kit #12030

When ordering five or more kits, please contact us for processing time and pricing at sales@cellsignal.com.

Kit Includes Volume Solution Color
Bcl-2 Rabbit mAb Coated Microwells 96 tests
Bcl-2 Mouse Detection mAb 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 Cross-Reactivity

H

Reactivity Key:  H=Human
Species enclosed in parentheses are predicted to react based on 100% sequence homology.

Description

The PathScan® Total Bcl-2 Sandwich ELISA Kit is a solid phase sandwich enzyme-linked immunosorbent assay (ELISA) that detects endogenous levels of Bcl-2. A Bcl-2 Rabbit mAb has been coated onto the microwells. After incubation with cell lysates, Bcl-2 protein is captured by the coated antibody. Following extensive washing, a Bcl-2 Mouse Detection mAb is added to detect the captured Bcl-2 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.Antibodies in kit are custom formulations specific to kit.

Specificity / Sensitivity

The PathScan® Total Bcl-2 Sandwich ELISA Kit recognizes endogenous levels of Bcl-2 protein 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.

ELISA

ELISA

Figure 2. The relationship between protein concentration of lysates from Jurkat cells, λ phosphatase-treated, untreated, or treated with Paclitaxel #9807 (1.0 mM, 20 hr) at 37ºC, and the absorbance at 450 nm is shown.

ELISA - Western correlation

ELISA - Western correlation

Figure 1. Treatment of Jurkat cells with Paclitaxel stimulates phosphorylation of Bcl-2 at Ser70, detected by the PathScan® Phospho-Bcl-2 (Ser70) Sandwich ELISA Kit #11874, but does not affect the levels of total Bcl-2 detected by PathScan® Total Bcl-2 Sandwich ELISA Kit #12030. Jurkat cells were λ phosphatase-treated, untreated, or treated with Paclitaxel #9807 (1.0 mM, 20 hr) at 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.

Background

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).

  1. Murphy, K.M. et al. (2000) Cell Death Differ 7, 102-11.
  2. Zhu, L. et al. (1999) J Biol Chem 274, 33267-73.
  3. Maundrell, K. et al. (1997) J Biol Chem 272, 25238-42.
  4. Yamamoto, K. et al. (1999) Mol Cell Biol 19, 8469-78.
  5. Ling, Y.H. et al. (1998) J Biol Chem 273, 18984-91.
  6. Huang, S.T. and Cidlowski, J.A. (2002) FASEB J 16, 825-32.
  7. Deng, X. et al. (2001) J Biol Chem 276, 23681-8.

Application References

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