MLLT4 Antibody

What is MLLT4 and why is it a significant target for antibody-based research?

MLLT4 (Myeloid/Lymphoid or Mixed-Lineage Leukemia; Translocated to, 4), also known as AF6 or Afadin, is a multi-domain protein involved in signaling and organization of cell junctions during embryogenesis. It has received significant attention due to its identification as a fusion partner of the ALL-1 gene in acute myeloid leukemias with t(6;11)(q27;q23) translocation .

MLLT4 has diverse cellular functions, including:

• Maintenance of adherens junctions between radial glial cells (RGCs) in cortical development

• Involvement in chromosomal translocations associated with leukemogenesis

• Role as a transcriptional coregulator in hematopoietic development

Its complex role in both normal development and disease states makes it an important target for antibody-based detection and characterization in various research contexts .

What applications are MLLT4 antibodies most commonly used for in research settings?

Based on the technical specifications of available antibodies, MLLT4 antibodies are most widely utilized in the following applications:

Researchers should note that MLLT4 antibodies show reactivity with human, mouse, and rat samples, making them versatile for comparative studies across species . The choice of application should be guided by the specific research question, with Western blotting being particularly useful for characterizing fusion proteins in leukemia research .

How should researchers validate the specificity of MLLT4 antibodies for experimental use?

Robust validation is essential to ensure experimental reliability when working with MLLT4 antibodies. A comprehensive validation protocol should include:

• Positive and negative control samples: Utilize cell lines known to express MLLT4 (e.g., HeLa cells) as positive controls and MLLT4-knockout or knockdown samples as negative controls .

• Multiple detection methods: Confirm antibody specificity using at least two independent techniques (e.g., Western blot combined with immunofluorescence).

• Peptide competition assay: Pre-incubate the antibody with immunizing peptide to confirm binding specificity through signal abolishment .

• Cross-reactivity assessment: Test against related proteins or in tissues where MLLT4 expression is well-characterized.

• Molecular weight verification: For Western blotting, confirm detection at the expected molecular weight (~208 kDa for full-length protein; different for fusion proteins) .

Research by multiple groups indicates that epitope-affinity purified antibodies offer superior specificity compared to conventional purification methods when detecting MLLT4 and its fusion variants .

What are the key considerations for selecting an appropriate MLLT4 antibody for leukemia research?

When studying MLLT4 in leukemia contexts, especially MLL-rearranged leukemias, researchers should consider these critical factors:

• Epitope location: Select antibodies targeting the appropriate domain based on your research focus. For MLL-AF4 fusion protein detection, antibodies recognizing the C-terminal domain of MLLT4 are preferable .

• Fusion protein specificity: Some antibodies may detect wild-type MLLT4 but fail to recognize fusion proteins or vice versa. Validation with positive controls expressing the specific fusion protein of interest is essential .

• Sensitivity requirements: For minimal residual disease (MRD) monitoring, highly sensitive antibodies are required, often necessitating supplementation with molecular techniques like RT-PCR for MLL-AF4 fusion detection .

• Cross-species reactivity: For translational research using mouse models of MLL leukemia, antibodies with confirmed reactivity in both human and mouse samples are advantageous .

• Application compatibility: Ensure the antibody is validated for your intended application, particularly for flow cytometry applications in leukemia phenotyping .

The literature suggests combining antibody detection with molecular methods for comprehensive characterization of MLLT4 fusion proteins in leukemia research settings .

How can researchers effectively use MLLT4 antibodies to study protein interactions in cell junction research?

MLLT4/Afadin plays a critical role in cell adhesion and junction formation. To effectively study these interactions:

• Co-immunoprecipitation protocols: For MLLT4 co-IP studies, use a buffer system containing 50mM Tris-HCl (pH 7.5), 150mM NaCl, 1% NP-40, and appropriate protease inhibitors. Pre-clear lysates with protein A/G beads before antibody incubation to reduce non-specific binding .

• Proximity ligation assays: When studying MLLT4 interactions with other adhesion molecules like N-cadherin, combine primary antibodies from different species with species-specific secondary antibodies for in situ proximity detection .

• Immunofluorescence co-localization: Use confocal microscopy with validated MLLT4 antibodies (typically at 1:100 dilution) in combination with antibodies against known interaction partners such as N-cadherin to visualize co-localization at cell junctions .

• Domain-specific antibodies: For mapping specific interaction domains, select antibodies targeting different regions of MLLT4, particularly those recognizing the PDZ domain which mediates many protein-protein interactions .

Research has demonstrated that MLLT4's interaction with RGCs is critical for controlling progenitor cell proliferation during neocortical development, making these methodological approaches particularly valuable for developmental neurobiology research .

What methodological approaches are recommended when using MLLT4 antibodies in mouse models of leukemia?

When utilizing mouse models to study MLLT4-related leukemias, several methodological considerations are critical:

• Model selection and validation: Different MLL-fusion protein mouse models recapitulate distinct aspects of human disease. For MLL-AF4 studies, conditional knockout models using Cre-lox systems (such as Mllt4-flox mice crossed with Emx1-Cre mice) allow tissue-specific expression .

• Antibody validation in mouse tissues: Confirm cross-reactivity and specificity of MLLT4 antibodies in mouse samples before experimental use. Western blotting of mouse tissues alongside human positive controls is recommended .

• Immunophenotyping protocols: For flow cytometry of mouse leukemia cells, use the following antibody panel alongside anti-MLLT4:

  • CD19 and CD33 to distinguish lymphoid versus myeloid lineage

  • CD34 and CD10 for assessing leukemia stem cell populations

  • B220 and CD43 for B-cell development stages

• Transplantation studies: When transplanting MLLT4 fusion-expressing cells into recipient mice (NSG or NSGS), confirm engraftment through peripheral blood sampling at 4-week intervals using species-specific MLLT4 antibodies .

Research has demonstrated that the cellular context significantly affects MLLT4 fusion protein activity, with MLL-AF4 showing stronger leukemogenic potential in lymphoid versus myeloid contexts .

How does long non-coding RNA MLLT4-AS1 interact with MLLT4, and what methodologies are appropriate for studying this relationship?

MLLT4-AS1 is a long non-coding RNA associated with MLLT4 that has emerged as an important regulatory molecule in autophagy and cancer. Research methodologies for investigating this relationship include:

• RNA-protein interaction studies: RNA fluorescence in situ hybridization (RNA-FISH) combined with MLLT4 immunofluorescence can visualize co-localization. This requires careful fixation protocols (typically 4% paraformaldehyde for 10 minutes) and RNase-free conditions .

• RNA immunoprecipitation (RIP): To detect physical interactions between MLLT4-AS1 and proteins:

  • Use formaldehyde cross-linking (1% for 10 minutes)

  • Immunoprecipitate with MLLT4 antibody

  • Extract RNA from precipitates and perform RT-PCR for MLLT4-AS1

• Expression correlation analysis: Quantify both MLLT4-AS1 and MLLT4 levels across tissue samples using RT-qPCR and Western blotting, respectively, to establish expression pattern relationships .

• Functional studies: The impact of MLLT4-AS1 on MLLT4 function can be assessed through:

  • MLLT4-AS1 overexpression/knockdown followed by MLLT4 protein analysis

  • Autophagy assessment using LC3-II/LC3-I ratios and SQSTM1/p62 levels

  • Evaluation of downstream targets like ATG14

Research has revealed that MLLT4-AS1 is upregulated by the MTORC inhibitor PP242 and rapamycin in cervical cells, promoting autophagy through interaction with myosin-9 protein and ATG14 transcription regulation rather than directly with MLLT4 protein itself .

What technical challenges exist in detecting MLLT4 fusion proteins in leukemia samples, and how can they be addressed?

Detection of MLLT4 fusion proteins presents several technical challenges that require specific methodological approaches:

• Protein size considerations: The MLL-AF4 fusion protein is large (~376 kDa), necessitating:

  • Special Western blotting conditions with gradient gels (3-8% Tris-acetate)

  • Extended transfer times (overnight at 30V, 4°C)

  • Low-concentration SDS (0.1%) in transfer buffer

• Epitope accessibility: Fusion proteins may have altered epitope exposure. Recommendations include:

  • Using antibodies targeting multiple epitopes

  • Testing different antigen retrieval methods for IHC (citrate buffer pH 6.0 vs. EDTA buffer pH 9.0)

  • Optimizing fixation protocols (shorter fixation times of 12-24 hours)

• Sample heterogeneity: Leukemia samples often contain mixed populations. Address by:

  • Flow cytometry sorting prior to protein analysis

  • Single-cell approaches when appropriate

  • Correlation with molecular genetic testing (RT-PCR for fusion transcripts)

• Low abundance detection: For minimal residual disease monitoring:

  • Combine immunological detection with molecular amplification methods

  • Consider using RT-PCR quantification of MLL-AF4 fusion as a complementary approach

  • Implement sensitive detection systems (ECL-Plus or fluorescent secondary antibodies)

Research indicates that MLL-AF4 is predominantly associated with pro-B-ALL and much more rarely with AML, making proper selection of control samples critical for antibody validation in different leukemia subtypes .

What are the optimal experimental conditions for studying MLLT4's role in cell adhesion and migration using antibody-based techniques?

MLLT4/Afadin's critical role in cell adhesion and migration requires specific experimental approaches:

• Cell junction visualization: For optimal immunofluorescence of MLLT4 at cell junctions:

  • Fix cells with 4% paraformaldehyde (10 min, room temperature)

  • Permeabilize with 0.2% Triton X-100 (5 min)

  • Block with 5% BSA for 1 hour

  • Incubate with MLLT4 antibody (typically 1:100 dilution) overnight at 4°C

  • Co-stain with junction markers (E-cadherin, ZO-1) for co-localization analysis

• Migration assay protocols: For studying MLLT4's impact on cell migration in cancer models:

  • Transwell chamber assays with 8μm pore size are most effective

  • Plate 2-5×10⁴ cells in serum-free medium in upper chamber

  • Use 10% FBS-containing medium in lower chamber as chemoattractant

  • After MLLT4 manipulation (overexpression/knockdown), fix and stain migrated cells after 24-48 hours

• Live-cell imaging: To track dynamic MLLT4 behavior:

  • Generate MLLT4-GFP fusion constructs for live visualization

  • Use spinning disk confocal microscopy with temperature and CO₂ control

  • Capture images every 5-10 minutes for up to 24 hours

• Wound healing assays: For studying collective cell migration:

  • Create wounds in confluent monolayers using 200μl pipette tips

  • Apply MLLT4 antibody to visualize protein recruitment to leading edges

  • Image at 0, 12, 24, and 48 hours post-wounding

Research has demonstrated that MLLT4-AS1 overexpression significantly inhibits the invasive and migratory abilities of cervical cancer cells, suggesting important roles for the MLLT4 regulatory network in cancer progression .

How can researchers effectively utilize MLLT4 antibodies in investigating neurodevelopmental processes and disorders?

MLLT4/Afadin plays a crucial role in neurodevelopment, particularly in cortical formation. When studying these processes:

• Brain section preparation: For optimal MLLT4 detection in brain tissue:

  • Fix embryonic or adult brain tissue with 4% paraformaldehyde (24h for embryonic, 48h for adult)

  • Cryoprotect in 30% sucrose before sectioning

  • Use 20μm sections for embryonic and 40μm for adult tissue

  • Perform heat-mediated antigen retrieval (citrate buffer, pH 6.0)

• Neuronal culture immunostaining: For primary neurons or neural progenitors:

  • Culture cells on poly-L-lysine coated coverslips

  • Fix with 4% paraformaldehyde (10 min, room temperature)

  • Block with 10% normal goat serum containing 0.1% Triton X-100

  • Co-stain with neuronal markers (Tbr2, Tuj1) and MLLT4 antibody (1:100 dilution)

• Cortical development analysis: When studying double cortex formation:

  • Use coronal sections at consistent anatomical levels

  • Perform immunostaining with layer-specific markers alongside MLLT4

  • Quantify cortical thickness and cell distribution across layers

  • Compare wildtype, heterozygous, and homozygous conditional knockouts

• Neurogenesis assays: For proliferation studies:

  • Pulse-label with BrdU (50mg/kg) 2 hours before sacrifice

  • Double-stain for BrdU and MLLT4 to assess proliferating populations

  • Quantify ventricular zone and subventricular zone progenitors

Research has shown that conditional inactivation of MLLT4 using Emx1-Cre mice results in widespread disruption of adherens junctions between radial glial cells and dramatically increased progenitor proliferation, leading to a double cortex-like phenotype in adult mice. This makes MLLT4 antibodies valuable tools for studying cortical malformation disorders .

What are the recommended protocols for using MLLT4 antibodies in conjunction with autophagy studies?

The emerging connection between MLLT4-AS1 and autophagy regulation necessitates specific methodological approaches:

• Autophagy flux assessment: When studying MLLT4-AS1's impact on autophagy:

  • Monitor LC3-II/LC3-I ratio by Western blot (use 1:1000 dilution of MLLT4 antibody)

  • Assess SQSTM1/p62 degradation as autophagy marker

  • Include Bafilomycin A1 (100nM, 4h) treatment to block autophagosome-lysosome fusion

  • Compare results with and without mTOR inhibitors (PP242 at 10μM or rapamycin)

• Co-localization studies: For assessing MLLT4-AS1's interaction with autophagy machinery:

  • Perform RNA-FISH for MLLT4-AS1 combined with immunofluorescence for ATG14

  • Use confocal microscopy with Z-stack acquisition (0.5μm steps)

  • Quantify co-localization using Pearson's correlation coefficient

• Immunoprecipitation protocol: For protein-RNA interactions:

  • Use crosslinking with 1% formaldehyde (10 min at room temperature)

  • Lyse cells in buffer containing 50mM Tris-HCl (pH 7.4), 150mM NaCl, 1mM EDTA, 1% NP-40

  • Immunoprecipitate with MYH-9 antibody

  • Extract RNA and perform RT-PCR for MLLT4-AS1

• In vivo autophagy visualization: For tissue samples from xenograft models:

  • Stain tumor sections with LC3 antibody to visualize autophagy puncta

  • Compare LC3-II levels and p62 levels in different treatment groups

  • Correlate with MLLT4-AS1 expression levels

Research has shown that MLLT4-AS1 was upregulated by H3K27ac modification with PP242 treatment, and knockdown of MLLT4-AS1 reversed autophagy by modulating ATG14 expression. This suggests a regulatory pathway where MLLT4-AS1 associates with myosin-9 protein to promote ATG14 transcription and induce autophagy .

How can researchers effectively develop antibody-drug conjugates targeting MLLT4-related proteins for therapeutic applications?

While MLLT4 itself has not been widely targeted for antibody-drug conjugate (ADC) development, related research on leukocyte immunoglobulin-like receptor B4 (LILRB4) in AML provides valuable methodological insights for researchers interested in developing MLLT4-targeting therapies:

• Antibody selection criteria: When selecting antibodies for potential ADC development:

  • Prioritize antibodies demonstrating high specificity and affinity (Kd < 10nM)

  • Confirm target expression patterns across healthy vs. diseased tissues

  • Select antibodies with demonstrated internalization capacity

  • Consider immunoglobulin isotype (IgG1 preferred for most ADC applications)

• Conjugation technologies: For creating homogeneous antibody-drug conjugates:

  • Microbial transglutaminase (MTGase)-mediated conjugation provides site-specific attachment

  • Diazidoamine branched linkers offer controllable drug-to-antibody ratios

  • Glutamic acid-valine-citrulline linkers provide stability in circulation with intracellular cleavability

• Payload selection considerations: When selecting cytotoxic payloads:

  • Antimitotic agents like monomethyl auristatin F (MMAF) are effective for hematological malignancies

  • DNA-damaging agents provide alternative mechanisms of action

  • Consider cell-permeable metabolites for potential bystander effects

• Pharmacokinetic assessment: For evaluating ADC behavior in vivo:

  • Measure plasma concentration over time using anti-human IgG ELISA

  • Assess drug-to-antibody ratio stability in circulation

  • Evaluate drug accumulation in tumor vs. healthy tissues

Research has demonstrated that homogeneous antibody-drug conjugates can achieve remarkable therapeutic effects with minimal toxicity in xenograft mouse models of disseminated human AML, suggesting similar approaches could be valuable for MLLT4-targeted therapies in leukemias with MLL rearrangements .