Cell Signaling Technology

Product Pathways - Tyrosine Kinase / Adaptors

PathScan® Phospho-Bcr-Abl (Tyr177) Sandwich ELISA Kit #7950

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Kit Includes Volume Solution Color
Abl Mouse mAb coated microwells 96 tests
Phospho-Bcr (Tyr177) Rabbit Detection Antibody 11 ml Green
Anti-rabbit 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

PathScan® Phospho-Bcr-Abl (Tyr177) Sandwich ELISA Kit is a solid phase sandwich enzyme-linked immunosorbent assay (ELISA) that detects endogenous levels of Bcr-Abl protein phosphorylated at Tyr177 of Bcr. A c-Abl mouse mAb has been coated on the microwells. After incubation with cell lysates, Bcr-Abl and c-Abl protein (phospho and nonphospho) is captured by the coated antibody. Following extensive washing, a phospho-Bcr (Tyr177) rabbit detection antibody is added to detect captured phospho-Bcr-Abl protein. Anti-rabbit IgG, HRP-linked antibody is then used to recognize the bound detection antibody. HRP substrate TMB is added to develop color. The magnitude of the absorbance for this developed color is proportional to the quantity of Bcr-Abl protein phosphorylated at Tyr177.Antibodies in kit are custom formulations specific to kit.

Specificity / Sensitivity

PathScan® Phospho-Bcr-Abl (Tyr177) Sandwich ELISA Kit #7950 detects endogenous levels of Bcr-Abl protein when phosphorylated at Tyr177 in human cells, as shown in Figure 1. The 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 - Western correlation

ELISA - Western correlation

Figure 1. Constitutive phosphorylation of Bcr-Abl in K-562 cells lysed in the presence of phosphatase inhibitors* (phospho lysate) is detected by PathScan® Phospho-Bcr-Abl (Tyr177) Sandwich ELISA Kit #7950. In contrast, a low level of phospho-Bcr-Abl protein is detected in K-562 cells lysed in the absence of phosphatase inhibitors* (nonphospho lysate). Absorbance at 450 nm is shown in the top figure while corresponding western blots using Bcr Antibody #3902 (left) and Phospho-Bcr (Tyr177) Antibody #3901 (right) are shown in the bottom figure. *Phosphatase inhibitors include sodium pyrophosphate, β-glycerophosphate, and Na3VO4.

Sensitivity

Sensitivity

Figure 2. The relationship between protein concentration of phospho or non-phospho lysates and the absorbance at 450 nm is shown. Unstarved K-562 cells were cultured (106 cells/ml) and lysed with or without addition of phosphatase inhibitor to the lysis buffer (phospho or non-phospho lysate, respectively).

Background

The Bcr gene was orginally identified by its presence in the chimeric Bcr-Abl oncogene (1). The amino-terminal region of Bcr contains an oligomerization domain, a serine/threonine kinase domain, and a region that binds SH2 domains. The middle of the protein has a PH domain and a region of sequence similarity to the guanine nucleotide exchange factors for the Rho family of GTP binding proteins. The carboxy-terminal region may be involved in a GTPase activating function for the small GTP-binding protein Rac (2,3). The function of wild type Bcr in cells remains unclear. PDGF receptor may use Bcr as a downstream signaling mediator (4). Research studies have shown that the Bcr-Abl fusion results in production of a constitutively active tyrosine kinase, which causes chronic myelogenous leukemia (CML) (5). Tyr177 of Bcr is phosphorylated in the Bcr-Abl fusion protein, which plays an important role in transforming the activity of Bcr-Abl (6). Phosphorylated Tyr177 provides a docking site for Gab2 and GRB2 (7,8).

  1. Groffen, J. et al. (1984) Cell 36, 93-99.
  2. Maru, Y. et al. (1991) Cell 67, 459-468.
  3. Che, W. et al. (2001) Circulation 104, 1399-1406.
  4. Abe, J. I. et al. (2001) Ann. N.Y. Acad. Sci. 947, 341-343.
  5. Voncken, J. W. et al. (1995) Cell 80, 719-728.
  6. He, Y. et al. (2002) Blood 99, 2957-2968.
  7. Sattler, M. et al. (2002) Cancer Cell 1, 479-492.
  8. Warmuth, M. et al. (1995) J. Biol. Chem. 272, 33260-33270.

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