Product Pathways - PathScan ELISA

PathScan^® Total Axl Sandwich ELISA Kit #7040

Kit Includes	Volume	Solution Color
Axl Mouse Antibody Coated Microwells	96 tests
Axl Rabbit Detection Antibody	11 milliliters	Green
Anti-rabbit IgG, HRP-linked Antibody	11 milliliters	Red
TMB Substrate	11 milliliters	Colorless
STOP Solution	11 milliliters	Colorless
Sealing Tape	2 sheets
20X Wash Buffer	25 milliliters	Colorless
Sample Diluent	25 milliliters	Blue
Cell Lysis Buffer (10X) # 9803	15 milliliters	Yellowish
Axl Mouse Monoclonal Antibody

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 (10X), which is stored at –20°C (packaged separately).

Species Cross-Reactivity

Reactivity Key: H=Human

Description

The PathScan^® Total Axl Sandwich ELISA Kit is a solid phase sandwich enzyme-linked immunosorbent assay (ELISA) that detects endogenous levels of total 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, an Axl rabbit antibody is added to detect captured 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 total Axl protein.

Specificity / Sensitivity

CST's PathScan^® Total Axl Sandwich ELISA Kit #7040 detects endogenous levels of total Axl protein in human cells, as shown in Figure 1. The kit sensitivity is shown in Figure 2.

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

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 recently characterized 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).

Application References

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Companion Products

This product is intended for research purposes only. The product is not intended to be used for therapeutic or diagnostic purposes in humans or animals.