Product Pathways - PathScan ELISA
PathScan® Phospho-FLT3 (Tyr591) Sandwich ELISA Kit #7206
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|Kit Includes||Volume||Solution Color|
|FLT3 Mouse mAb coated microwells||96 tests|
|Phospho-FLT3 (Tyr591) 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|
|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 enclosed in parentheses are predicted to react based on 100% sequence homology.
CST's PathScan® Phospho-FLT3 (Tyr591) Sandwich ELISA Kit is a solid phase sandwich enzyme-linked immunosorbent assay (ELISA) that detects endogenous levels of FLT3 protein phosphorylated at Tyr591. A FLT3 Mouse mAb has been coated onto the microwells. After incubation with cell lysates, FLT3 (phospho or nonphospho) protein is captured by the coated antibody. Following extensive washing, a phospho-FLT3 (Tyr 591) Rabbit Antibody is added to detect the captured phospho-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 FLT3 phosphorylated at Tyr591.Antibodies in kit are custom formulations specific to kit.
Specificity / Sensitivity
PathScan® Phospho-FLT3 (Tyr591) Sandwich ELISA Kit #7206 detects endogenous levels of FLT3 protein phosphorylated at Tyr591 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
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.
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.
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- 3461 Phospho-FLT3 (Tyr591) Antibody
- 3462 FLT3 (8F2) Rabbit mAb
- 7202 PathScan® Total FLT3 Sandwich ELISA Kit
- 7074 Anti-rabbit IgG, HRP-linked Antibody
- 9803 Cell Lysis Buffer (10X)
- 9808 Phosphate Buffered Saline (PBS-20X)
- 9809 Phosphate Buffered Saline with Tween 20 (PBST-20X)
- 9998 BSA
- 7004 TMB Substrate
- 7002 STOP Solution
US Patent No. 7,183,385 and foreign equivalents assigned to Cell Signaling Technology, Inc.
For Research Use Only. Not For Use In Diagnostic Procedures.