Cell Signaling Technology

Product Pathways - PathScan ELISA

PathScan® Phospho-FLT3 (panTyr) Sandwich ELISA Kit #7761

When ordering five or more kits, please contact us for processing time and pricing at sales@cellsignal.com.

Important Ordering Details: Product is assembled upon order to ensure maximum activity. Domestic: Please allow up to two weeks for your order to be processed and shipped. International: Please allow up to three weeks, depending on the country, for your order to be processed and shipped.

Kit Includes Volume Solution Color
FLT3 Rabbit mAb coated microwells 96 tests
Phospho-Tyrosine Mouse Detection Antibody 11 ml Green
Anti-mouse 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
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

CST's PathScan® Phospho-FLT3 (panTyr) Sandwich ELISA Kit is a solid phase sandwich enzyme-linked immunosorbent assay (ELISA) that detects endogenous levels of tyrosine-phosphorylated FLT3 protein. A FLT3 rabbit antibody has been coated on the microwells. After incubation with cell lysates, FLT3 protein (phospho and nonphospho) is captured by the coated antibody. Following extensive washing, a phospho-tyrosine mouse detection antibody is added to detect captured tyrosine-phosphorylated FLT3 protein. Anti-mouse 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 FLT3 protein phosphorylated on tyrosine.Antibodies in kit are custom formulations specific to kit.

Specificity / Sensitivity

PathScan® Phospho-FLT3 (panTyr) Sandwich ELISA Kit #7761 detects endogenous levels of FLT3 protein phosphorylated at Tyr residues 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 phosphorylated of FLT3 in SEM cells is detected by PathScan® Phospho-FLT3 (panTyr) Sandwich ELISA kit #7761. 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

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 (10-6 cells/ml) were left untreated 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).

  1. Shurin, M.R. et al. (1998) Cytokine Growth Factor Rev. 9, 37-48.
  2. Naoe, T. et al. (2001) Cancer Chemother. Pharmacol. 48 Suppl1, S27-S30.
  3. Namikawa, R. et al. (1996) Stem Cells 14, 388-395.
  4. Beslu, N. et al. (1996) J. Biol. Chem. 271, 20075-20081.
  5. Zhang, S. and Broxmeyer, H.E. (2000) Biochem. Biophys. Res. Commun. 277, 195-199.
  6. Zhang, S. et al. (1999) J. Leukoc. Biol. 65, 372-380.
  7. Mizuki, M. et al. (2000) Blood 96, 3907-3914.

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