FTL3 Antibody, HRP conjugated

What is FLT3 and what is its role in normal hematopoiesis?

FLT3 (FMS-like tyrosine kinase 3) is a class III receptor tyrosine kinase that plays a critical role in regulating hematopoiesis. It consists of an extracellular Ig-like domain, a transmembrane domain, a juxtamembrane domain, and a tyrosine kinase domain. FLT3 is exclusively expressed in hematopoietic progenitors and functions in normal cell development through several mechanisms .

Upon binding of the FLT3 ligand (FL) dimer, FLT3 dimerizes and transphosphorylates multiple tyrosine residues on the tyrosine kinase domain. This phosphorylation recruits SH2- or PTB-containing adaptors and subsequently phosphorylates downstream signaling proteins, including AKT, MAPK, STAT5, and SFK family members. These signals collectively regulate anti-apoptosis, cell survival, and proliferation pathways crucial for normal hematopoietic development .

What molecular alterations of FLT3 are significant in hematological malignancies?

FLT3 mutations represent one of the most common genetic alterations in acute myeloid leukemia (AML), occurring in approximately 30-40% of adult patients . Two primary types of activating FLT3 mutations have significant research and clinical importance:

• Internal tandem duplication (ITD) mutations: Present in about 20% of AML cases, these mutations involve duplications within the juxtamembrane domain that disrupt the autoinhibitory function, leading to constitutive activation of the receptor . FLT3-ITD mutations significantly alter cellular behavior compared to ligand-activated wild-type FLT3 and are associated with poor prognosis .

• Point mutations: These primarily occur in the activation loop of the tyrosine kinase domain, particularly at the D835 residue.

Mechanistically, these mutations result in constitutive autophosphorylation of FLT3, which is sufficient to mediate factor-independent proliferation and survival . The aberrant activation particularly affects the immature form of the receptor, which shows constitutive phosphorylation even when the mature form retains ligand-dependent activation patterns .

What are the key characteristics of FLT3 antibodies, particularly HRP conjugated variants?

FLT3 antibodies, including HRP (horseradish peroxidase) conjugated versions, are essential tools for detecting and studying FLT3 expression and activation. The key characteristics include:

• Recognition specificity: High-quality FLT3 antibodies specifically target epitopes within the FLT3 protein structure. For example, some antibodies recognize regions within amino acids 701-800 of human FLT3 (NP_004110.2) .

• Molecular weight detection: FLT3 typically appears at 130kDa/160kDa in its observed form, while the calculated molecular weight is 113kDa . This discrepancy is due to post-translational modifications, particularly glycosylation.

• HRP conjugation advantage: Direct HRP conjugation eliminates the need for secondary antibody incubation steps, reducing background and increasing specificity in applications like ELISA and Western blot .

• Storage requirements: Typically stored at -20°C with 50% glycerol to prevent freeze-thaw damage .

• Buffer composition: Usually preserved in buffers containing components like Proclin 300, glycerol, and PBS at pH 7.4 to maintain stability and activity .

What are the validated applications for FLT3 antibody, HRP conjugated in leukemia research?

FLT3 antibody, HRP conjugated, has several validated applications in leukemia research workflows:

• ELISA (Enzyme-Linked Immunosorbent Assay): The primary application for HRP-conjugated FLT3 antibodies is in ELISA systems, where they enable direct detection of FLT3 protein without secondary antibody requirements . This is particularly valuable when quantifying FLT3 expression levels across patient samples or cell lines.

• Western Blot Analysis: For detecting FLT3 expression and activation status in cell lysates. The expected molecular weight ranges from 130-160kDa depending on glycosylation status and mutations . HRP conjugation allows for direct chemiluminescence detection following primary antibody binding.

• Immunoprecipitation coupled with phosphorylation analysis: FLT3 antibodies can be used to immunoprecipitate the receptor from cell lysates, followed by phosphotyrosine detection to assess activation status . This approach is essential when studying signaling pathway activation downstream of FLT3.

• Immunohistochemistry on paraffin-embedded tissues: For detecting FLT3 expression in bone marrow biopsies and other relevant tissues . Dilution ratios of 1:50 to 1:200 are typically recommended for immunohistochemical applications.

What is the recommended protocol for detecting FLT3 autophosphorylation using HRP-conjugated antibodies?

The detection of FLT3 autophosphorylation requires a carefully optimized protocol:

• Cell preparation:

  • Culture FLT3-expressing cells (e.g., AML cell lines like MV-4-11 or THP-1)

  • Starve cells from serum and growth factors for 12 hours in medium containing 0.5% FCS

  • Stimulate with FLT3 ligand (100 ng/mL) for 10 minutes at 37°C or leave unstimulated for basal phosphorylation assessment

• Lysis procedure:

  • Wash cells with ice-cold PBS

  • Lyse with buffer containing: 50 mmol/L HEPES (pH 7.4), 10% glycerol, 150 mmol/L NaCl, 1% Triton X-100, 1 mmol/L EDTA, 1 mmol/L EGTA, protease inhibitors, and 1 mmol/L sodium orthovanadate

  • Clarify lysates by centrifugation at 20,000 g for 20 minutes

• Immunoprecipitation:

  • Incubate lysates with FLT3 antibody

  • Add Protein A/G-Plus-Sepharose to capture antibody-antigen complexes

  • Wash immunoprecipitates three times with lysis buffer

• Western blot detection:

  • Separate proteins by SDS-PAGE

  • Transfer to PVDF membrane

  • Block with appropriate blocking buffer

  • Probe with anti-phosphotyrosine antibody or phospho-specific antibodies

  • For direct detection: use HRP-conjugated FLT3 antibody (1:100 - 1:500 dilution)

  • Develop using chemiluminescence detection system (e.g., ECL-Plus)

How can FLT3 antibody be used to distinguish between wild-type and mutant FLT3 in research samples?

Distinguishing between wild-type and mutant FLT3 requires specific approaches:

• Western blot pattern analysis:

  • Wild-type FLT3: Shows predominantly the mature glycosylated form (160 kDa) with ligand-dependent phosphorylation

  • FLT3-ITD: Shows altered ratio between mature and immature forms, with constitutive phosphorylation of the lower molecular weight immature form (130 kDa)

• Phosphorylation kinetics:

  • Wild-type FLT3: Requires ligand stimulation for phosphorylation

  • FLT3-ITD: Shows constitutive phosphorylation that may be enhanced by ligand stimulation

• Competition assay:

  • Pre-incubate cells with anti-FLT3 IgG polyclonal antibody (10 μg/mL) for 2 hours

  • Add FLT3-targeted compounds (e.g., FL-DM1 at 20 nM)

  • Incubate for 48 hours before measuring cell viability

  • This approach helps determine FLT3-specific targeting versus off-target effects

• Downstream signaling analysis:

  • FLT3-ITD mutations show characteristic patterns of STAT5 activation not typically seen with ligand-activated wild-type FLT3

  • Monitoring phosphorylation of Erk1/2 and Akt can differentiate between mutant and wild-type signaling pathways

How can FLT3 antibody be incorporated into drug development workflows for AML therapeutics?

FLT3 antibodies serve critical functions in developing targeted therapies for AML:

• Target validation:

  • FLT3 antibodies confirm expression levels in patient samples and cell lines

  • Western blot and immunohistochemistry with FLT3 antibodies establish expression in >90% of AML blasts, validating FLT3 as a widespread therapeutic target

• Drug conjugate development:

  • FLT3 antibodies or FLT3 ligand can be conjugated to cytotoxic agents (e.g., emtansine/DM1) using linkers like SPDP (N-succinimidyl 3-(2-pyridyldithio)propionate)

  • For conjugation validation, FLT3 antibodies confirm successful creation of conjugates through size shift analysis

• Target engagement studies:

  • FLT3 antibodies in competition assays determine whether novel compounds engage the intended target

  • For example, pre-incubation with anti-FLT3 IgG (10 μg/mL) followed by FL-DM1 treatment helps establish specificity of targeting

• Efficacy assessment:

  • Monitoring changes in FLT3 phosphorylation status after drug treatment using phospho-specific antibodies

  • Comparing effects on mutant versus wild-type FLT3 to establish therapeutic window

• Patient stratification biomarker development:

  • FLT3 antibodies aid in developing assays to identify patients most likely to benefit from FLT3-targeted therapies

  • Distinguishing between FLT3-mutant and FLT3-wild type expression patterns through immunoblotting

What methods exist for analyzing FLT3 signaling pathways using antibody-based approaches?

Comprehensive analysis of FLT3 signaling requires sophisticated antibody-based methodologies:

• Phosphoprotein profiling:

  • Immunoblotting with phospho-specific antibodies against key signaling nodes:

    • p-Erk1/2 (Tyr204): For MAPK pathway activation

    • p-Akt (Ser473): For PI3K pathway activation

    • p-STAT5: For JAK/STAT pathway activation

  • Comparison between ligand-stimulated and basal conditions to differentiate constitutive activation

• Signaling pathway dissection:

  • Sequential immunoprecipitation with FLT3 antibody followed by probing for interacting partners

  • Immunoblotting for adaptor proteins recruited to activated FLT3 (e.g., SH2- or PTB-containing adaptors)

• Kinetic analysis of pathway activation:

  • Time-course experiments with FLT3 ligand stimulation (0-60 minutes)

  • Fixation and antibody-based detection of phosphorylation events at different time points

  • Particularly valuable for comparing wild-type versus mutant FLT3 signaling dynamics

• Inhibitor studies combined with antibody detection:

  • Treatment with pathway-specific inhibitors (MEK, PI3K, JAK inhibitors)

  • Western blot analysis with FLT3 antibodies and phospho-specific antibodies

  • Determines which pathways are essential for FLT3-mediated effects in different cellular contexts

How can FLT3 antibodies be used to evaluate emerging therapeutic approaches like antibody-drug conjugates?

FLT3 antibodies are invaluable tools for evaluating novel therapeutic modalities targeting FLT3:

• Antibody-drug conjugate (ADC) development assessment:

  • Characterizing binding specificity of conjugated versus unconjugated antibodies

  • Evaluating internalization kinetics through fluorescently labeled FLT3 antibodies

  • Measuring drug-to-antibody ratio through comparative analysis

• Evaluation of FL-based drug conjugates:

  • FL-DM1 conjugates show selective cytotoxicity toward FLT3-expressing cells

  • IC50 values: 12.9 nM for THP-1 cells and 1.1 nM for MV-4-11 cells

  • FLT3 antibodies confirm target specificity through competition assays

• Mechanism of action studies:

  • Cell cycle analysis: FL-DM1 induces G2/M arrest, detectable through flow cytometry after PI staining

  • Apoptosis assessment: Detection of caspase-3 activation through Western blot or flow cytometry

  • FLT3 antibodies confirm target engagement prior to downstream effects

• Selectivity verification:

  • FLT3 antibodies demonstrate selective targeting of FLT3-expressing cells versus non-expressing controls

  • For example, FL-DM1 selectively targets HCD-57 cells transformed by FLT3-ITD but not parental HCD-57 cells lacking FLT3 expression

What are common technical challenges when using FLT3 antibodies and how can they be addressed?

Researchers frequently encounter several technical challenges when working with FLT3 antibodies:

• Variable glycosylation patterns:

  • Challenge: FLT3 exists in multiple glycosylated forms (130kDa/160kDa observed vs. 113kDa calculated) , leading to diffuse or multiple bands.

  • Solution: Use positive controls with known FLT3 expression patterns. Consider enzymatic deglycosylation (PNGase F treatment) to confirm band identity when necessary.

• Cross-reactivity concerns:

  • Challenge: Some FLT3 antibodies may cross-react with related receptor tyrosine kinases.

  • Solution: Validate antibody specificity using FLT3-knockout or knockdown samples. For HRP-conjugated antibodies, optimize antibody concentration (typically 1:100 - 1:500 for Western blot) to minimize non-specific binding.

• Phosphorylation-dependent epitope masking:

  • Challenge: Phosphorylation status may affect antibody recognition of certain epitopes.

  • Solution: Use multiple antibodies recognizing different epitopes. When studying phosphorylation, first immunoprecipitate with a total FLT3 antibody, then probe with phospho-specific antibodies.

• Receptor internalization effects:

  • Challenge: Ligand stimulation induces receptor internalization, potentially affecting detection.

  • Solution: Include time-course experiments and consider subcellular fractionation to track receptor localization during signaling studies.

• Competition assay optimization:

  • Challenge: Achieving proper blocking without non-specific effects.

  • Solution: Use isotype control antibodies alongside anti-FLT3 blocking. Optimize pre-incubation time (typically 2 hours) before adding test compounds .

How should researchers interpret different band patterns when analyzing FLT3 using Western blot?

Interpreting FLT3 Western blot patterns requires understanding several key aspects:

• Normal pattern in wild-type FLT3-expressing cells:

  • Two predominant bands: mature fully glycosylated form (~160 kDa) and immature partially glycosylated form (~130 kDa)

  • Upon ligand stimulation, phosphorylation occurs primarily in the mature form

  • Ratio between mature and immature forms is typically consistent in wild-type cells

• FLT3-ITD mutation patterns:

  • Altered ratio between mature and immature forms, often with stronger immature band

  • Constitutive phosphorylation of the immature form even without ligand

  • The mature form may retain ligand-dependent phosphorylation patterns

• Degradation patterns:

  • Multiple bands below 130 kDa may indicate proteolytic degradation

  • Fresh sample preparation and inclusion of appropriate protease inhibitors can minimize this issue

• Interpretation table for common patterns:

What controls are essential when conducting FLT3 antibody-based experiments?

Rigorous control strategies are critical for reliable FLT3 antibody experiments:

• Positive controls:

  • Cell lines with validated FLT3 expression: THP-1, MV-4-11 (FLT3-ITD positive)

  • Recombinant FLT3 protein standards for quantitative assays

  • Stimulation control: Cells treated with FLT3 ligand (100 ng/mL for 10 minutes)

• Negative controls:

  • Cell lines lacking FLT3 expression (e.g., HCD-57 parental cells)

  • Isotype-matched irrelevant antibody controls for immunoprecipitation

  • For HRP-conjugated antibodies: matched isotype control antibody with similar HRP conjugation

• Specificity controls:

  • Peptide competition: Pre-incubation of antibody with immunizing peptide should abolish specific signal

  • Genetic knockdown/knockout: siRNA or CRISPR-modified cells with reduced/absent FLT3 expression

  • For drug development: Competition with anti-FLT3 IgG (10 μg/mL) to confirm target specificity

• Technical controls:

  • Loading controls: Probing for housekeeping proteins (β-actin, GAPDH) to ensure equal loading

  • Phosphorylation controls: Serum stimulation as a positive control for general phosphorylation pathways

  • For HRP-conjugated antibodies: Include secondary antibody-only controls to assess non-specific binding

How might FLT3 antibodies contribute to developing next-generation AML therapeutics?

FLT3 antibodies hold significant potential for advancing AML treatment through several emerging approaches:

What methodological innovations might enhance the utility of FLT3 antibodies in future research?

Several technological advances could significantly expand the research applications of FLT3 antibodies:

• Single-cell phosphoproteomic integration:

  • Combining FLT3 antibodies with mass cytometry (CyTOF) or single-cell Western approaches

  • This would allow analysis of FLT3 signaling heterogeneity within AML cell populations

  • Could reveal distinct responder vs. non-responder subpopulations within a single patient sample

• Live-cell imaging applications:

  • Development of non-interfering FLT3 antibody fragments conjugated to fluorescent proteins

  • Would enable real-time tracking of FLT3 localization, internalization, and trafficking

  • Could assess how different FLT3 mutations affect receptor dynamics in living cells

• Proximity labeling approaches:

  • FLT3 antibodies fused to promiscuous biotin ligases (BioID or TurboID)

  • Would enable unbiased mapping of the FLT3 interactome under different conditions

  • Could identify novel therapeutic targets in the FLT3 signaling network

• Nanobody development:

  • Engineering smaller FLT3-targeting antibody fragments with improved tissue penetration

  • These could enhance in vivo imaging of FLT3-expressing leukemia and potentially improve therapeutic delivery

  • Would expand applications beyond traditional antibody limitations

• Patient-derived organoid compatibility:

  • Optimizing FLT3 antibody protocols for 3D culture systems

  • Would enable assessment of FLT3 targeting in more physiologically relevant models

  • Could bridge the gap between cell line studies and clinical applications

How can FLT3 antibodies support personalized medicine approaches in AML treatment?

FLT3 antibodies can drive personalized medicine in AML through several applications:

• Multiparameter patient stratification:

  • Beyond FLT3 mutation status, antibody-based profiling can assess:

    • Total FLT3 expression levels

    • Phosphorylation status of multiple downstream effectors

    • Association with other signaling proteins

  • This could identify patients likely to respond to specific FLT3-targeted therapies despite mutation status

• Ex vivo drug sensitivity testing:

  • FLT3 antibodies can evaluate on-target activity in patient samples exposed to various FLT3 inhibitors

  • Correlating drug responses with FLT3 signaling changes could predict clinical efficacy

  • HRP-conjugated antibodies would facilitate rapid assessment in limited patient material

• Minimal residual disease monitoring:

  • Developing highly sensitive flow cytometry or immunoassay methods using FLT3 antibodies

  • Could detect residual leukemic cells with aberrant FLT3 expression or signaling

  • Would enable personalized adjustment of therapy intensity based on MRD status

• Combination therapy rational design:

  • FLT3 antibody-based signaling analysis could identify patient-specific bypass pathways

  • These insights would inform rational combinations tailored to individual resistance mechanisms

  • Would move beyond "one-size-fits-all" approaches to FLT3-targeted therapy