Cell Signaling Technology

Product Pathways - PathScan ELISA

PathScan® Phospho-Axl (panTyr) Sandwich ELISA Kit #7042

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Kit Includes Volume Solution Color
Axl Mouse Ab Coated Microwells 96 tests
Phospho-Tyrosine 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

The PathScan® Phospho-Axl (panTyr) Sandwich ELISA Kit is a solid phase sandwich enzyme-linked immunosorbent assay (ELISA) that detects endogenous levels of tyrosine-phosphorylated Axl protein. An Axl mouse antibody has been coated on the microwells. After incubation with cell lysates, Axl protein (phospho and nonphospho) is captured by the coated antibody. Following extensive washing, a phospho-tyrosine rabbit antibody is added to detect captured tyrosine-phosphorylated Axl 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 Axl protein phosphorylated on tyrosine residues.Antibodies in kit are custom formulations specific to kit.

Specificity / Sensitivity

CST's PathScan® Phospho-Axl (panTyr) Sandwich ELISA Kit #7042 detects endogenous levels of tyrosine-phosphorylated Axl protein 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 Axl in NCI-H2347 cells lysed in the presence of phosphatase inhibitors* (phospho lysate) is detected by PathScan® Phospho-Axl (panTyr) Sandwich ELISA Kit #7042 (top, right). In contrast, a low level of phospho-Axl protein is detected in NCI-H2347 cells lysed in the absence of phosphatase inhibitors* (nonphospho lysate). Similar levels of total Axl protein from both nonphospho or phospho lysates are detected by PathScan® Total Axl Sandwich ELISA Kit #7040 (top, left). Absorbance at 450 nm is shown in the top figure while corresponding western blots using a phospho-Axl (Tyr814) rabbit antibody (right) or a total Axl (C44G1) Rabbit mAb #4566 (left) 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 nonphospho lysates and the absorbance at 450 nm is shown. NCI-H2347 cells were cultured (85% confluence) and lysed with or without the addition of phosphatase inhibitor to the lysis buffer (phospho or nonphospho lysate).

Background

Axl, Sky, and Mer are three members of a receptor tyrosine kinase (RTK) family that share a conserved intracellular tyrosine kinase domain and an extracellular domain similar to those seen in cell adhesion molecules. These RTKs bind the vitamin K-dependent protein growth-arrest-specific 6 (Gas6), which is structurally related to the protein S anticoagulation factor (1). Upon binding to its receptor, Gas6 activates phosphatidylinositol 3-kinase (PI3K) and its downstream targets Akt and S6K, as well as NF-κB (2,3). A large body of evidence supports a role for Gas6/Axl signaling in cell growth and survival in normal and cancer cells (4).

  1. Crosier, K.E. and Crosier, P.S. (1997) Pathology 29, 131-135.
  2. Demarchi, F. et al. (2001) J. Biol. Chem. 276, 31738-31744.
  3. Lee, W. P. et al. (2002) Oncogene 21, 329-336.
  4. Bellosta, P. et al. (1997) Oncogene 15, 2387-2397.

Application References

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