Product Pathways - Tyrosine Kinase / Adaptors
PathScan® Phospho-c-Abl (Tyr412) Sandwich ELISA Kit #12070
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|Kit Includes||Volume||Solution Color|
|Abl Mouse mAb coated microwells||96 tests|
|Phospho-c-Abl (Tyr412) Rabbit Detection Ab||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 enclosed in parentheses are predicted to react based on 100% sequence homology.
PathScan® Phospho-c-Abl (Tyr412) 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 Mouse mAb 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-c-Abl (Tyr412) Rabbit Detection Antibody is added to detect phospho-Bcr-Abl and phospho-c-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 absorbance for the developed color is proportional to the quantity of Bcr-Abl or c-Abl protein phosphorylated at Tyr412.Antibodies in kit are custom formulations specific to kit.
Specificity / Sensitivity
PathScan® Phospho-c-Abl (Tyr412) Sandwich ELISA Kit #12070 recognizes endogenous levels of Bcr-Abl or c-Abl protein when phosphorylated at Tyr412 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
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 (Tyr412) Sandwich ELISA Kit #12070. 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 panel) and Phospho-c-Abl (Tyr412) (247C7) Rabbit mAb #2865 (right panel) 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 nonphospho 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 inhibitors to the lysis buffer (phospho or nonphospho lysate, respectively).
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).
- Wang, J.Y. et al. (2000) Oncogene 19, 5643-5650.
- Van Etten, R.A. et al. (1999) Trends Cell. Biol. 9, 179-182.
- Danial, N.N. et al. (2000) Oncogene 19, 2523-2531.
- Shaul, Y. et al. (2000) Cell Death Differ. 7, 10-16.
- Brasher, B.B. et al. (2000) J. Biol. Chem. 275, 35631-35637.
- Pluk, H. et al. (2002) Cell 108, 247-259.
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- 7950 PathScan® Phospho-Bcr-Abl (Tyr177) Sandwich ELISA Kit
- 7903 PathScan® Phospho-c-Abl (panTyr) Sandwich ELISA Kit
- 3901 Phospho-Bcr (Tyr177) Antibody
- 3902 Bcr Antibody
- 2865 Phospho-c-Abl (Tyr412) (247C7) Rabbit mAb
- 2862 c-Abl Antibody
- 9803 Cell Lysis Buffer (10X)
- 9808 Phosphate Buffered Saline (PBS-20X)
- 9809 Phosphate Buffered Saline with Tween 20 (PBST-20X)
- 9998 BSA
- 7004 TMB Substrate
- 7002 STOP Solution
For Research Use Only. Not For Use In Diagnostic Procedures.