Key Product Details

Species Reactivity

Validated:

Human

Cited:

Human, Mouse

Applications

Validated:

Flow Cytometry, CyTOF-ready

Cited:

Western Blot, Immunocytochemistry, Immunoprecipitation

Label

Unconjugated

Antibody Source

Monoclonal Mouse IgG1 Clone # 66903
View all Flt-3/Flk-2 Primary Antibodies »

Product Specifications

Immunogen

Mouse myeloma cell line NS0-derived recombinant human Flt‑3/Flk‑2
Asn27-Asn541
Accession # AAA18947

Specificity

Detect human Flt‑3/Flk‑2 in direct ELISAs. In direct ELISAs, no cross-reactivity with recombinant mouse Flt-3, recombinant human (rh) SCF R, rhPDGF R alpha, or rhPDGF R beta is observed.

Clonality

Monoclonal

Host

Mouse

Isotype

IgG1

Scientific Data Images for Human Flt‑3/Flk‑2 Antibody

Detection of Flt‑3/Flk‑2 in THP-1 cells by Flow Cytometry.

THP-1 cells were stained with Mouse Anti-Human Flt‑3/Flk‑2 Monoclonal Antibody (Catalog # MAB812, filled histogram) or isotype control antibody (Catalog # MAB002, open histogram), followed by Phycoerythrin-conjugated Anti-Mouse IgG Secondary Antibody (Catalog # F0102B). View our protocol for Staining Membrane-associated Proteins.

Detection of Flt‑3/Flk‑2 in PBMC monocytes by Flow Cytometry.

PBMC monocytes were stained with Mouse Anti-Human Flt‑3/Flk‑2 Monoclonal Antibody (Catalog # MAB812, filled histogram) or isotype control antibody (Catalog # MAB002, open histogram), followed by Phycoerythrin-conjugated Anti-Mouse IgG Secondary Antibody (Catalog # F0102B). View our protocol for Staining Membrane-associated Proteins.
Detection of Human Flt-3/Flk-2 by Immunocytochemistry/ Immunofluorescence

Detection of Human Flt-3/Flk-2 by Immunocytochemistry/ Immunofluorescence

FLT3-ITD mislocalizes to the perinuclear region in AML cells. (a) Schematic representations of wild-type FLT3 (FLT3-wt) and an FLT3 internal tandem duplication (FLT3-ITD) mutant showing the extracellular domain (ECD, blue), the transmembrane domain (TM, yellow), the kinase domain (pink), and the ITD (green). (b) Fixed THP-1, RS4-11, MV4-11, MOLM-14, or Kasumi-6 cells were permeabilized and subsequently immunostained with anti-FLT3 ECD antibody. Arrowheads indicate the perinuclear region. Bars, 10 µm. Note that FLT3-wt localized to the plasma membrane, whereas FLT3-ITD accumulated in the perinuclear region. Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/34811450), licensed under a CC-BY license. Not internally tested by R&D Systems.
Detection of Human Flt-3/Flk-2 by Western Blot

Detection of Human Flt-3/Flk-2 by Western Blot

FLT3-ITD retention in the Golgi region is dependent on its tyrosine kinase activity. (a, b) MOLM-14 cells were treated for 4 h with AC220 (a) or PKC412 (b). Lysates were immunoblotted for FLT3, phospho-FLT3 Tyr842 (pFLT3Y842), AKT, pAKT, ERK, pERK, STAT5, and pSTAT5. Full length blots are presented in Supplementary Fig. 5. (c) MOLM-14 cells were treated with AC220 (upper graph) or PKC412 (lower graph) for 48 h. Cell proliferation was assessed by ATP production. Results are means ± s.d. (n = 3). (d) Lysates from MOLM-14 were treated with peptide N-glycosidase F (PNGase F) or endoglycosidase H (endo H) then immunoblotted with anti-FLT3 antibody. CG complex-glycosylated form, HM high mannose form, DG deglycosylated form. Full length blots are presented in Supplementary Fig. 5. (e, f) MOLM-14 cells were treated with 10 nM AC220 or 100 nM PKC412 for 8 h (e) or 16 h (f). (e) Fixed cells were permeabilized, then immunostained with anti-FLT3 (red) and anti-calnexin (ER marker, green). Insets show the magnified images of the boxed area. Bars, 10 µm. (f) Non-permeabilized cells were immunostained with an anti-FLT3 extracellular domain (ECD) antibody. Bars, 10 µm. Note that FLT3 tyrosine kinase inhibitors inactivated FLT3, then released the receptor from the Golgi region for localization to the PM. Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/34811450), licensed under a CC-BY license. Not internally tested by R&D Systems.
Detection of Human Flt-3/Flk-2 by Immunocytochemistry/ Immunofluorescence

Detection of Human Flt-3/Flk-2 by Immunocytochemistry/ Immunofluorescence

FLT3-ITD retention in the Golgi region is dependent on its tyrosine kinase activity. (a, b) MOLM-14 cells were treated for 4 h with AC220 (a) or PKC412 (b). Lysates were immunoblotted for FLT3, phospho-FLT3 Tyr842 (pFLT3Y842), AKT, pAKT, ERK, pERK, STAT5, and pSTAT5. Full length blots are presented in Supplementary Fig. 5. (c) MOLM-14 cells were treated with AC220 (upper graph) or PKC412 (lower graph) for 48 h. Cell proliferation was assessed by ATP production. Results are means ± s.d. (n = 3). (d) Lysates from MOLM-14 were treated with peptide N-glycosidase F (PNGase F) or endoglycosidase H (endo H) then immunoblotted with anti-FLT3 antibody. CG complex-glycosylated form, HM high mannose form, DG deglycosylated form. Full length blots are presented in Supplementary Fig. 5. (e, f) MOLM-14 cells were treated with 10 nM AC220 or 100 nM PKC412 for 8 h (e) or 16 h (f). (e) Fixed cells were permeabilized, then immunostained with anti-FLT3 (red) and anti-calnexin (ER marker, green). Insets show the magnified images of the boxed area. Bars, 10 µm. (f) Non-permeabilized cells were immunostained with an anti-FLT3 extracellular domain (ECD) antibody. Bars, 10 µm. Note that FLT3 tyrosine kinase inhibitors inactivated FLT3, then released the receptor from the Golgi region for localization to the PM. Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/34811450), licensed under a CC-BY license. Not internally tested by R&D Systems.
Detection of Human Flt-3/Flk-2 by Immunocytochemistry/ Immunofluorescence

Detection of Human Flt-3/Flk-2 by Immunocytochemistry/ Immunofluorescence

In AML cells, FLT3-ITD can activate AKT, ERK, and STAT5 before it reaches the PM. (a–c) MOLM-14 (a, b), MV4-11, or Kasumi-6 cells (c) were treated with inhibitors of ER export (BFA or M-COPA) for 8 h. (a) MOLM-14 cells treated with 1 µM BFA (middle panels) or 1 µM M-COPA (bottom panels) were stained with anti-FLT3 (red) and calnexin (ER marker, green). Insets show the magnified images of the boxed area. Bars, 10 µm. (b) Lysates were immunoblotted for FLT3, phospho-FLT3 Tyr842 (pFLT3Y842), AKT, pAKT, ERK, pERK, STAT5, and pSTAT5. To examine pFLT3Y591, FLT3 was immunoprecipitated, then immunoblotted. (c) MV4-11 (left) or Kasumi-6 cells (right) were treated with M-COPA for 8 h, then immunoblotted. Full length blots are presented in Supplementary Fig. 5. Note that BFA and M-COPA inhibited the activation of AKT and ERK but not that of STAT5 through blocking FLT3-ITD trafficking from the ER to the Golgi apparatus. (d, e) MOLM-14 cells were treated with monensin (inhibitor of Golgi export) for 8 h. (d) Cells treated with 100 nM monensin were stained with anti-FLT3 (red) and lectin-HPA (Golgi marker, blue). Dashed line, cell border. Bars, 10 µm. (e) Lysates were immunoblotted with the indicated antibody. To examine pFLT3Y591, FLT3 was immunoprecipitated, then immunoblotted. Full length blots are presented in Supplementary Fig. 5. (f) MV4-11 (left) or Kasumi-6 cells (right) were treated with monensin for 8 h, then immunoblotted. Full length blots are presented in Supplementary Fig. 6. Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/34811450), licensed under a CC-BY license. Not internally tested by R&D Systems.
Detection of Human Flt-3/Flk-2 by Immunocytochemistry/ Immunofluorescence

Detection of Human Flt-3/Flk-2 by Immunocytochemistry/ Immunofluorescence

In AML cells, FLT3-ITD can activate AKT, ERK, and STAT5 before it reaches the PM. (a–c) MOLM-14 (a, b), MV4-11, or Kasumi-6 cells (c) were treated with inhibitors of ER export (BFA or M-COPA) for 8 h. (a) MOLM-14 cells treated with 1 µM BFA (middle panels) or 1 µM M-COPA (bottom panels) were stained with anti-FLT3 (red) and calnexin (ER marker, green). Insets show the magnified images of the boxed area. Bars, 10 µm. (b) Lysates were immunoblotted for FLT3, phospho-FLT3 Tyr842 (pFLT3Y842), AKT, pAKT, ERK, pERK, STAT5, and pSTAT5. To examine pFLT3Y591, FLT3 was immunoprecipitated, then immunoblotted. (c) MV4-11 (left) or Kasumi-6 cells (right) were treated with M-COPA for 8 h, then immunoblotted. Full length blots are presented in Supplementary Fig. 5. Note that BFA and M-COPA inhibited the activation of AKT and ERK but not that of STAT5 through blocking FLT3-ITD trafficking from the ER to the Golgi apparatus. (d, e) MOLM-14 cells were treated with monensin (inhibitor of Golgi export) for 8 h. (d) Cells treated with 100 nM monensin were stained with anti-FLT3 (red) and lectin-HPA (Golgi marker, blue). Dashed line, cell border. Bars, 10 µm. (e) Lysates were immunoblotted with the indicated antibody. To examine pFLT3Y591, FLT3 was immunoprecipitated, then immunoblotted. Full length blots are presented in Supplementary Fig. 5. (f) MV4-11 (left) or Kasumi-6 cells (right) were treated with monensin for 8 h, then immunoblotted. Full length blots are presented in Supplementary Fig. 6. Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/34811450), licensed under a CC-BY license. Not internally tested by R&D Systems.

Applications for Human Flt‑3/Flk‑2 Antibody

Application
Recommended Usage

CyTOF-ready

Ready to be labeled using established conjugation methods. No BSA or other carrier proteins that could interfere with conjugation.

Flow Cytometry

0.25 µg/106 cells
Sample: THP-1 and PBMC monocytes

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Flow Cytometry

Formulation, Preparation, and Storage

Purification

Protein A or G purified from ascites

Reconstitution

Reconstitute at 0.5 mg/mL in sterile PBS. For liquid material, refer to CoA for concentration.

Formulation

Lyophilized from a 0.2 μm filtered solution in PBS with Trehalose. *Small pack size (SP) is supplied either lyophilized or as a 0.2 µm filtered solution in PBS.

Shipping

Lyophilized product is shipped at ambient temperature. Liquid small pack size (-SP) is shipped with polar packs. Upon receipt, store immediately at the temperature recommended below.

Stability & Storage

Use a manual defrost freezer and avoid repeated freeze-thaw cycles.
  • 12 months from date of receipt, -20 to -70 °C as supplied.
  • 1 month, 2 to 8 °C under sterile conditions after reconstitution.
  • 6 months, -20 to -70 °C under sterile conditions after reconstitution.

Calculators

The reconstitution calculator allows you to quickly calculate the volume of a reagent to reconstitute your vial. Simply enter the mass of reagent and the target concentration and the calculator will determine the rest.

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Background: Flt-3/Flk-2

The Flt-3 (fms-like tyrosine kinase) receptor, also named Flk-2 (fetal liver kinase) and Stk-1(stem cell tyrosine kinase) is a member of the class III subfamily of receptor tyrosine kinases that also includes KIT, the receptor for SCF and FMS, the receptor for M-CSF. The extracellular region of these receptors contains five immunoglobulin-like domains and the intracellular region contains a split kinase domain. Human Flt-3 cDNA encodes a 993 amino acid (aa) residue type I membrane protein with a 26 aa residue signal peptide, a 515 aa extracellular domain with 10 potential N-linked glycosylation sites, a 21 aa residue transmembrane domain and a 431 aa residue cytoplasmic domain. Mouse Flt-3 has also been cloned and shown to share 85% amino acid sequence identity with human Flt-3. Flt-3 expression has been detected in various tissues, including placenta, gonads, and tissues of nervous and hematopoietic origin. Among hematopoietic cells, the expression of Flt-3 was found to be restricted to the highly enriched stem/progenitor cell populations. The ligand for Flt-3 (FL) has been identified to be a transmembrane protein with structural homology to M-CSF and SCF. Recombinant soluble Flt-3/Fc chimeric protein has been shown to bind FL with high affinity and is a potent FL antagonist.

References

  1. Rosnet, O. et al. (1996) Acta. Haemato. 95:218.
  2. Drexler, H.G. (1996) Leukemia 10:588.

Long Name

fms-like Tyrosine Kinase 3

Alternate Names

CD135, Flk-2, Flt3, STK-1

Entrez Gene IDs

2322 (Human); 14255 (Mouse)

Gene Symbol

FLT3

UniProt

AAA18947

Additional Flt-3/Flk-2 Products

Product Documents for Human Flt‑3/Flk‑2 Antibody

Certificate of Analysis

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Product Specific Notices for Human Flt‑3/Flk‑2 Antibody

For research use only

Citations for Human Flt‑3/Flk‑2 Antibody

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