Product Pathways - PathScan ELISA
PathScan® Total FLT3 Sandwich ELISA Kit #7202
| Kit Includes | Volume | Solution Color |
|---|---|---|
| FLT3 Mouse mAb coated microwells | 96 tests | |
| FLT3 Detection Ab | 11 milliliters | Green |
| Anti-rabbit IgG HRP-Linked Ab | 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 |
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
H
Reactivity Key: H=Human
Description
CST's PathScan® Total FLT3 Sandwich ELISA Kit is a solid phase sandwich enzyme-linked immunosorbent assay (ELISA) that detects endogenous levels of total FLT3 protein. A FLT3 Mouse mAb #4584* has been coated onto the microwells. After incubation with cell lysates, both phospho- and nonphospho-FLT3 proteins are captured by the coated antibody. Following extensive washing, FLT3 Rabbit mAb #4583* is added to detect both the captured phospho- and nonphospho-FLT3 protein. Anti-rabbit IgG, HRP-linked Antibody #7074* is then used to recognize the bound detection antibody. HRP substrate, TMB, is added to develop color. The magnitude of optical density for this developed color is proportional to the quantity of total FLT3 protein.* Antibodies in kit are custom formulations specific to kit.
Specificity / Sensitivity
CST's PathScan® Total FLT3 Sandwich ELISA Kit #7202 detects endogenous levels of total FLT3 Protein. As shown in Figure 1, a high level of phosphorylated FLT3 (Tyr591) is detected in SEM cells where FLT3 is constitutively phosphorylated, and the levels are significantly reduced in SEM cells treated with CT-53518, an inhibitor of FLT3 tyrosine phosphorylation. The levels of total FLT3 (phospho and non-phospho) protein detected by PathScan® Total FLT3 Sandwich ELISA Kit #7202 remain unchanged. Phospho-FLT3 (Tyr591) in MV4;11 or RS4;11 cells also can be detected by this kit (data not shown).
Sandwich ELISA
Figure 1: FLT3 is constitutively phosphorylated in SEM cells, detected by PathScan® Phospho-FLT3 (Tyr591) Sandwich ELISA Kit #7206. In contrast, only a low level of phospho-FLT3 protein is detected in SEM cells treated with CT-53518, an inhibitor of FLT3 tyrosine phosphorylation. The inhibitor does not affect the level of total FLT3 protein detected by PathScan® Total FLT3 Sandwich ELISA Kit #7202. OD 450 readings are shown in the top figure, while the corresponding Western blots using Phospho-FLT3 (Tyr591) Antibody #3461 (right panel) or FLT3 Rabbit mAb #3462 (left panel), are shown in the bottom figure.
Sandwich ELISA
Figure 2: The relationship between protein concentration of lysates from untreated and inhibitor-treated SEM cells and kit assay optical density readings. Unstarved, SEM cells cultured (10 6 cells/ml) were treated with CT-53518 (50 ng/ml) for 120 min at 37?C, and then lysed.
Sandwich ELISA
Figure 3: Kit specificity demonstrated by Western blot analysis of the ELISA-well captured protein is shown. Lysates were prepared from human SEM cells and incubated in wells coated with capture antibody #4584. Wells were then washed, and captured protein was solubilized in SDS gel loading buffer. SEM lysate (lane 1) and captured protein (lane 2) were analyzed by Western blot using FLT3 Antibody #4583. Two bands corresponding to the FLT3 protein are detected in the captured material (lane 2).
Background
FMS-related tyrosine kinase 3 (FLT3, also called Flk2), is a member of the type III receptor tyrosine kinase family, which includes c-Kit, PDGFR and M-CSF receptors. FLT3 is expressed on early hematopoietic progenitor cells and supports growth and differentiation within the hematopoietic system (1,2). FLT3 is activated after binding with its ligand FL, which results in a cascade of tyrosine autophosphorylation and tyrosine phosphorylation of downstream targets (3). The p85 subunit of PI3 kinase, SHP2, GRB2 and Shc are associated with FLT3 after FL stimulation (4-6). Tyr589/591 is located in the juxtamembrane region of FLT3 and may play an important role in regulation of FLT3 tyrosine kinase activity. Somatic mutations of FLT3 consisting of internal tandem duplications (ITDs) occur in 20% of patients with acute myeloid leukemia (7).
- Shurin, M.R. et al. (1998) Cytokine Growth Factor Rev. 9, 37-48.
- Naoe, T. et al. (2001) Cancer Chemother. Pharmacol. 48 Suppl1, S27-S30.
- Namikawa, R. et al. (1996) Stem Cells 14, 388-395.
- Beslu, N. et al. (1996) J. Biol. Chem. 271, 20075-20081.
- Zhang, S. and Broxmeyer, H.E. (2000) Biochem. Biophys. Res. Commun. 277, 195-199.
- Zhang, S. et al. (1999) J. Leukoc. Biol. 65, 372-380.
- Mizuki, M. et al. (2000) Blood 96, 3907-3914.
Application References
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