Product Pathways - Tyrosine Kinase / Adaptors
PathScan® Phospho-c-Abl (Tyr412) Sandwich ELISA Kit #12070
|Product Includes||Volume||Solution Color|
|ELISA Sample Diluent||25 ml||Blue|
|Anti-rabbit IgG, HRP-linked Antibody (ELISA Formulated)||1 ea||Red (Lyophilized)|
|Detection Antibody Diluent||11 ml||Green|
|HRP Diluent||11 ml||Red|
|Phospho-c-Abl (Tyr412) Rabbit Detection mAb||1 ea||Green (Lyophilized)|
|STOP Solution||11 ml||Colorless|
|TMB Substrate #7004||11 ml||Colorless|
|ELISA Wash Buffer (20X)||25 ml||Colorless|
|Cell Lysis Buffer (10X) #9803||15 ml||Yellowish|
|Sealing Tape||2 sheets|
|Abl Mouse mAb Coated Microwells||96 tests|
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.
如图1，PathScan® Phospho-c-Abl (Tyr412) Sandwich ELISA试剂盒#12070可以识别人细胞中Tyr412被磷酸化的Bcr-Abl或c-Abl蛋白。试剂盒的敏感性如图2所示。经内部测试确认，本试剂盒可以检测到所列物种中的蛋白。但对其他物种中的同源蛋白可能也可以检测。
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.
PathScan® Phospho-c-Abl (Tyr412) Sandwich ELISA是通过固相夹心酶联免疫吸附法（ELISA）来检测内源性的酪氨酸磷酸化的Bcr-Abl和c-Abl蛋白的水平。先将c-Abl Mouse mAb包被在微孔板上。与细胞裂解物孵育后，Bcr-Abl和c-Abl 便可被包被的抗体捕获。清洗彻底后，加入Phospho-c-Abl (Tyr412)兔单抗检测phospho-Bcr-Abl和phospho-c-Abl 的水平。再加入Anti-Rabbit IgG, HRP偶联抗体作为二抗来识别之前绑定的检测抗体。最后加入HRP底物—— TMB，进行显色。产生的吸光值与Tyr412磷酸化的Bcr-Abl和c-Abl蛋白的数量成正比。
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).图2. 磷酸化及未磷酸化的胞裂解液蛋白浓度和450nm处吸光度的关系如图所示。未饥饿处理的K-562细胞培养(106 cells/ml)且被裂解，加入或不加入磷酸化抑制剂（磷酸化或磷酸化）。
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.图1. K-562细胞裂解后持续磷酸化的Bcr-Abl和c-Abl在phosphatase inhibitors* (phospho lysate)存在的条件下，使用PathScan® Phospho-c-Abl (Tyr412) Sandwich ELISA Kit #12070进行检测。相对的，K-562细胞中低水平的phospho-Bcr-Abl和phospho-c-Abl在phosphatase inhibitors* (nonphospho lysate)存在的条件下也能被检测到。上图中展示了450nm处的吸光值，使用c-Abl抗体#2862（左）和Phospho-c-Abl (Tyr412) (247C7)兔mAb#2865（右）进行western blot分析的结果在下图中。
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).
c-Abl原癌基因编码一个非受体蛋白酪氨酸激酶，该激酶在多细胞动物中普遍表达且高度保守。c-Abl蛋白分布于胞核以及胞质中，参与调节细胞增殖、分化、凋亡细胞粘附和应激反应(1-3)。在体内，通过整合素激活、PDGF刺激以及结合到c-Jun、Nck 和RFX1 蛋白上(2,4)等各种生理刺激，能使c-Abl激酶的活力提高。调节c-Abl蛋白激酶活力的体内机制目前尚不完全清楚。Tyr245位点位于SH2和催化区之间的连接区域，Abl蛋白家族在这个位点处都是保守的。Tyr245位点的磷酸化涉及到c-Abl激酶的活化（5）。另外，位于Abl蛋白的激酶活化环处的Tyr412位点，对激酶的活性也是必需的（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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- 2862 c-Abl Antibody
- 2865 Phospho-c-Abl (Tyr412) (247C7) Rabbit mAb
- 3901 Phospho-Bcr (Tyr177) Antibody
- 3902 Bcr Antibody
- 7004 TMB Substrate
- 7903 PathScan® Phospho-c-Abl (panTyr) Sandwich ELISA Kit
- 7950 PathScan® Phospho-Bcr-Abl (Tyr177) Sandwich ELISA Kit
- 9803 Cell Lysis Buffer (10X)
- 9808 Phosphate Buffered Saline (PBS-20X)
- 9809 Phosphate Buffered Saline with Tween® 20 (PBST-20X)
- 9998 BSA
For Research Use Only. Not For Use In Diagnostic Procedures.
Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
PathScan is a trademark of Cell Signaling Technology, Inc.
Cell Signaling Technology® is a trademark of Cell Signaling Technology, Inc.
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