Product Pathways - PathScan ELISA

PathScan^® Total FLT3 Sandwich ELISA Kit #7202

Kit Includes	Volume	Solution Color
FLT3 Mouse mAb coated microwells	96 tests
Anti-rabbit IgG HRP-Linked Ab	11 milliliters	Red
TMB Substrate	11 milliliters	Colorless
STOP Solution	11 milliliters	Colorless
Sealing Tape	2 sheets
Wash Buffer (20X)	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

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 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 Antibody is added to detect both the captured phospho- and nonphospho-FLT3 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 FLT3 protein.

Specificity / Sensitivity

PathScan^®Total FLT3 Sandwich ELISA Kit #7202 detects endogenous levels of total FLT3 protein in human cells, as shown in Figure 1. The kit sensitivity is shown in Figure 2.

ELISA - Western correlation

Figure 1: Constitutive phosphorylated of FLT3 in SEM cells is 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. Absorbance at 450 nm is 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), is shown in the bottom figure.

Sensitivity

Figure 2: The relationship between total protein concentration of the lysate from untreated or inhibitor-treated SEM cells and absorbance at 450 nm is shown. Unstarved, SEM cells (106 cells/ml) were either not treated or treated with CT-53518 (50 ng/ml) for 120 min and then lysed.

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).

Application References

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Protocols

7202:: Sandwich ELISA

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.