Figure 1. Constitutive phosphorylation of Bcr-Abl and c-Abl in K-562 cells lysed in the presence of phosphatase inhibitors* (phospho lysate) is detected by PathScan® Phospho-c-Abl (panTyr) Sandwich ELISA Kit #7903. In contrast, a low level of phospho-Bcr-Abl and phospho-c-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 c-Abl Antibody #2862 (left) and Phospho-c-Abl (Tyr412) (247C7) Rabbit mAb #2865 (right) are shown in the bottom figure. *Phosphatase inhibitors include sodium pyrophosphate, β-glycerophosphate, and Na3VO4.
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).
|7903C||1 Kit (96 assays)||N/A|
|Product Includes||Volume||Solution Color|
|c-Abl Rabbit Antibody Coated Microwells||96 tests|
|Phospho Tyrosine Mouse Detection mAb||1 ea||Green (Lyophilized)|
|Anti-mouse IgG, HRP-linked Antibody (ELISA Formulated)||1 ea||Red (Lyophilized)|
|Detection Antibody Diluent||11 ml||Green|
|HRP Diluent||11 ml||Red|
|TMB Substrate 7004||11 ml|
|STOP Solution 7002||11 ml|
|Sealing Tape||2 ea|
|ELISA Wash Buffer (20X) 9801||25 ml|
|ELISA Sample Diluent||25 ml||Blue|
|Cell Lysis Buffer (10X) 9803||15 ml|
PathScan® Phospho-c-Abl (panTyr) Sandwich ELISA Kit is a solid phase sandwich enzyme-linked immunosorbent assay (ELISA) that detects endogenous levels of tyrosine-phosphorylated Bcr-Abl and c-Abl proteins. A c-Abl rabbit antibody has been coated on the microwells. After incubation with cell lysates, Bcr-Abl and c-Abl protein (phospho and nonphospho) are captured by the coated antibody. Following extensive washing, a phospho-tyrosine mouse detection antibody is added to detect captured tyrosine-phosphorylated Bcr-Abl and c-Abl 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 the absorbance for this developed color is proportional to the quantity of tyrosine-phosphorylated Bcr-Abl and c-Abl protein.
Antibodies in kit are custom formulations specific to kit.
NOTE: Prepare solutions with purified water.
*NOTE: Some PathScan® ELISA Kits may include HRP-Linked Streptavidin in place of HRP-Linked Antibody.
NOTE: Initial color of positive reaction is blue, which changes to yellow upon addition of STOP Solution.
posted November 2013
Protocol Id: 204
PathScan® Phospho-c-Abl (panTyr) Sandwich ELISA Kit #7903 detects endogenous levels of Bcr-Abl or c-Abl protein when phosphorylated at Tyr residues 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.
The c-Abl proto-oncogene encodes a nonreceptor protein tyrosine kinase that is ubiquitously expressed and highly conserved in metazoan evolution. c-Abl protein is distributed in both the nucleus and the cytoplasm of cells. It is implicated in regulating cell proliferation, differentiation, apoptosis, cell adhesion, and stress responses (1-3). c-Abl kinase activity is increased in vivo by diverse physiological stimuli including integrin activation; PDGF stimulation; and binding to c-Jun, Nck, and RFX1 (2,4). The in vivo mechanism for regulation of c-Abl kinase activity is not completely understood. Tyr245 is located in the linker region between the SH2 and catalytic domains. This positioning is conserved among Abl family members. Phosphorylation at Tyr245 is involved in the activation of c-Abl kinase (5). In addition, phosphorylation at Tyr412, which is located in the kinase activation loop of c-Abl, is required for kinase activity (6).
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