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.Learn more about how we get our images
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.Learn more about how we get our images
|Product Includes||Volume||Solution Color|
|Bcl-2 Rabbit mAb Coated Microwells||96 tests|
|Bcl-2 Mouse Detection mAb||11 ml||Green|
|TMB Substrate 7004||11 ml||Colorless|
|STOP Solution 7002||11 ml||Colorless|
|Sealing Tape||2 ea|
|ELISA Wash Buffer (20X)||25 ml||Colorless|
|ELISA Sample Diluent||25 ml||Blue|
|Cell Lysis Buffer (10X) 9803||15 ml||Yellowish|
NOTE: Refer to product-specific datasheets or product webpage for assay incubation temperature.
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.
1X Cell Lysis Buffer: 10X Cell Lysis Buffer (#9803): To prepare 10 ml of 1X Cell Lysis Buffer, add 1 ml of 10X Cell Lysis Buffer to 9 ml of dH2O, mix. Buffer can be stored at 4°C for short-term use (1–2 weeks).
Recommended: Add 1 mM phenylmethylsulfonyl fluoride (PMSF) (#8553) immediately before use.
NOTE: Refer to product-specific datasheet or webpage for lysis buffer recommendation.
Add 100 µl of STOP solution to each well. Shake gently for a few seconds.
NOTE: Initial color of positive reaction is blue, which changes to yellow upon addition of STOP solution.
posted June 2005
revised November 2013
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.
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.
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).
Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc. PathScan is a trademark of Cell Signaling Technology, Inc.
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