MLLT10 Antibody

What is MLLT10 and what are its key structural features?

MLLT10, also known as AF10 (ALL1-fused gene from chromosome 10 protein), is a transcriptional regulator protein encoded by the MLLT10 gene. It contains several key structural domains:

• N-terminal zinc finger domain

• C-terminal leucine zipper domain (OM-LZ)

• Octapeptide motif

• PHD finger-like domain

The leucine zipper motif is particularly significant as it mediates interactions with GAS41, which in turn interacts with integrase interactor-1 (INI1), a component of the SWI/SNF chromatin remodeling complex. This suggests MLLT10's involvement in chromatin modification and transcriptional regulation .

What is the normal biological function of MLLT10 in cells?

MLLT10 functions as a transcriptional regulator with several identified roles:

• Acts as a cofactor for DOT1L-mediated histone H3K79 methylation

• Regulates cell identity maintenance in somatic cells

• Functions in chromatin remodeling through interactions with the SWI/SNF complex

• May play a role in transcriptional initiation

Mechanistically, MLLT10 facilitates higher-order (di- and tri-) methylation of H3K79 through direct interaction with the histone methyltransferase DOT1L. This epigenetic modification is crucial for maintaining cell identity and preventing cellular reprogramming .

How is MLLT10 involved in hematological malignancies?

MLLT10 is implicated in several hematological malignancies through chromosomal translocations:

The resulting fusion proteins consistently retain the leucine zipper motif of MLLT10, which is believed to interfere with normal gene regulation through interactions with chromatin remodeling complexes. In a recent Chinese cohort study, patients with PICALM-MLLT10 fusion gene (median age 25 years) demonstrated poor survival, with three out of six patients dying within one year despite intensive treatment .

What is the role of MLLT10 in solid tumors?

Beyond hematological malignancies, MLLT10 has been implicated in several solid tumors:

• Colorectal cancer (CRC): MLLT10 promotes tumor migration, invasion, and metastasis via epithelial-mesenchymal transition (EMT). Higher expression of MLLT10 is associated with increased CRC cell migration and invasion both in vitro and in vivo. At metastatic sites, MLLT10 expression correlates with increased Vimentin and decreased E-Cadherin expression, indicating its role in EMT regulation .

• Non-small cell lung cancer (NSCLC): MLLT10 is targeted by miR-331-3p, which acts as a tumor suppressor. Overexpression of MLLT10 promotes tumor cell proliferation, EMT-mediated metastasis, and invasion in NSCLC. The miR-331-3p/MLLT10 axis has been proposed as a potential therapeutic target for NSCLC .

What experimental applications are MLLT10 antibodies suitable for?

MLLT10 antibodies have been validated for multiple experimental applications:

These applications allow researchers to detect endogenous levels of MLLT10/AF-10 protein in various experimental settings .

How can I optimize MLLT10 antibody use for detecting fusion proteins?

Detecting MLLT10 fusion proteins (such as PICALM-MLLT10 or MLL-MLLT10) requires special considerations:

• Epitope selection: Choose antibodies raised against regions preserved in the fusion protein. For MLL-MLLT10 fusions, antibodies targeting the C-terminal region of MLLT10 are preferable since this region is typically retained in the fusion.

• Size verification: Fusion proteins will have different molecular weights than wild-type MLLT10. For example:

  • Wild-type MLLT10: ~110-120 kDa

  • MLL-MLLT10: ~200-230 kDa (depending on breakpoints)

  • PICALM-MLLT10: ~140-170 kDa

• Dual antibody approach: For definitive identification, use antibodies against both fusion partners simultaneously (e.g., anti-MLL and anti-MLLT10) in co-immunoprecipitation or co-localization studies .

How can MLLT10 antibodies be used to study the role of MLLT10 in cellular reprogramming?

MLLT10 has been identified as a barrier to cellular reprogramming through its regulation of H3K79 methylation. To investigate this function:

• Chromatin immunoprecipitation (ChIP): Use MLLT10 antibodies in conjunction with H3K79me2/me3 antibodies to identify genomic regions where MLLT10 regulates H3K79 methylation during reprogramming.

• Co-immunoprecipitation (Co-IP): Implement MLLT10 antibodies to pull down associated proteins like DOT1L to understand the dynamic changes in complex formation during reprogramming.

• Loss-of-function/rescue experiments: After MLLT10 knockdown, perform rescue experiments using wild-type or mutant MLLT10 (particularly the DOT1L binding-impaired OM-LZ deletion mutant), and use antibodies to verify:

  • MLLT10 expression levels

  • H3K79 methylation status

  • Expression of pluripotency markers (OCT4, NANOG, SSEA4)

A key finding from research is that while wild-type MLLT10, when reintroduced, can reduce reprogramming efficiency in MLLT10 knockout cells, the DOT1L binding-impaired mutant cannot, suggesting that the MLLT10-DOT1L interaction is critical for maintaining somatic cell identity .

What technical considerations are important when validating MLLT10 antibody specificity?

Validating antibody specificity is crucial for reliable results:

• Positive and negative controls:

  • Positive: Cell lines with known MLLT10 expression (e.g., leukemia cell lines)

  • Negative: MLLT10 knockout cells generated via CRISPR/Cas9 (targeting splice site exon 2 or exon 3)

• Multiple detection methods:

  • Western blot should show a band at the expected molecular weight (~110-120 kDa)

  • Immunofluorescence should demonstrate proper nuclear localization

  • Signal should diminish with siRNA or CRISPR/Cas9-mediated MLLT10 knockdown

• Cross-reactivity assessment:

  • Test against related proteins (e.g., other MLL fusion partners)

  • Validate across multiple species if working with non-human models

• Epitope verification:

  • Use antibodies targeting different epitopes of MLLT10 to confirm results

  • Consider the specific MLLT10 isoform recognized by your antibody .

What are the optimal conditions for immunoprecipitation of MLLT10 protein complexes?

Successful immunoprecipitation of MLLT10 and its protein complexes requires specific conditions:

• Lysis buffer composition:

  • RIPA buffer with reduced detergent concentration to preserve protein-protein interactions

  • Include protease inhibitors, phosphatase inhibitors, and HDAC inhibitors

  • For chromatin-associated complexes, include DNase I treatment

• Antibody selection and protocol:

  • Use 2-5 μg of antibody per sample

  • Pre-clear lysates with protein A/G beads

  • Incubate antibody with lysate overnight at 4°C

  • For weaker interactions (like with DOT1L), consider crosslinking with DSP or formaldehyde

• Verification of interacting partners:

  • Key known interactors include DOT1L, GAS41, and components of the SWI/SNF complex

  • Western blot analysis should confirm these interactions

  • Consider mass spectrometry for unbiased identification of novel interactors .

How can MLLT10 antibodies be used to analyze the epigenetic function of MLLT10?

MLLT10's role in epigenetic regulation can be studied using several antibody-dependent approaches:

• Sequential ChIP (ChIP-reChIP):

  • First ChIP with anti-MLLT10 followed by second ChIP with anti-H3K79me2/me3

  • This identifies genomic regions where MLLT10 and H3K79 methylation co-occur

• Proximity-based labeling:

  • BioID approach using MLLT10 fusion with biotin ligase

  • Identify proximal proteins using streptavidin pulldown

  • Verify interactions with MLLT10 antibodies

• Functional domain analysis:

  • Compare wild-type MLLT10 with mutants (L107A histone-binding mutant or OM-LZ DOT1L-binding deletion mutant)

  • Use antibodies to confirm expression and localization

  • Assess effects on H3K79 methylation levels

How do I address potential cross-reactivity with MLLT10 fusion proteins in patient samples?

When analyzing patient samples that may contain MLLT10 fusion proteins:

• Use multiple antibodies targeting different regions:

  • Antibodies against N-terminal regions will detect only wild-type MLLT10

  • Antibodies against C-terminal regions will detect both wild-type and most fusion proteins

• Size discrimination:

  • Run gradient gels (4-15%) to better separate proteins of different molecular weights

  • Use size markers that span the range of both wild-type and fusion proteins

• Fusion-specific detection:

  • Design PCR primers spanning the fusion breakpoint

  • Use fluorescence in situ hybridization (FISH) as a complementary approach

  • Consider RNA-seq to identify fusion transcripts

• Controls for validation:

  • Include known positive controls (cell lines with specific MLLT10 fusions)

  • Include wildtype controls to establish baseline MLLT10 detection .

What are common pitfalls when analyzing MLLT10 expression across different cell types and tissues?

Researchers should be aware of several challenges:

• Expression level variations:

  • MLLT10 expression varies significantly across tissue types

  • Low expression may require signal amplification techniques

  • Consider enrichment of nuclear fraction before analysis

• Isoform diversity:

  • Multiple MLLT10 isoforms exist (including types I, III, and IV)

  • Antibodies may have differential affinity for specific isoforms

  • Western blots may show multiple bands representing different isoforms

• Subcellular localization:

  • MLLT10 primarily shows nuclear localization

  • Mutant forms may show aberrant localization patterns

  • Perform proper nuclear/cytoplasmic fractionation for accurate assessment

• Context-dependent interactions:

  • MLLT10's interaction partners may vary by cell type

  • Co-IP results should be interpreted in a cell-type specific context

  • Use cell-type appropriate positive controls .

How can MLLT10 antibodies be utilized in therapeutic target identification and validation?

MLLT10 antibodies can support therapeutic development through several approaches:

• Target validation in preclinical models:

  • Immunohistochemistry to confirm MLLT10 expression in patient-derived xenografts

  • Correlation of MLLT10 levels with response to DOT1L inhibitors

  • Analysis of MLLT10-dependent pathways in drug-resistant samples

• Biomarker development:

  • Quantitative assessment of MLLT10 levels in patient samples

  • Correlation with clinical outcomes and treatment response

  • Development of companion diagnostics for targeted therapies

• Mechanism-of-action studies:

  • Evaluation of drug effects on MLLT10-DOT1L interaction

  • Assessment of downstream epigenetic changes upon treatment

  • Identification of resistance mechanisms through altered MLLT10 function or localization .

What emerging technologies can enhance MLLT10 antibody-based research?

Several cutting-edge technologies offer promising avenues for MLLT10 research:

• Single-cell protein analysis:

  • Mass cytometry (CyTOF) with MLLT10 antibodies

  • Single-cell Western blotting

  • Co-detection of MLLT10 with other markers at single-cell resolution

• Advanced imaging approaches:

  • Super-resolution microscopy to visualize MLLT10 chromatin interactions

  • Live-cell imaging with fluorescently labeled antibody fragments

  • Multiplexed ion beam imaging (MIBI) for spatial analysis in tissues

• Proteomics integration:

  • Combine MLLT10 antibody-based purification with mass spectrometry

  • Phospho-proteomics to identify signaling pathways affected by MLLT10

  • Thermal proteome profiling to identify drugs targeting MLLT10 complexes

• CRISPR screens with antibody readouts:

  • Use MLLT10 antibodies as readouts for CRISPR screens

  • Identify genes that modulate MLLT10 expression or localization

  • Combine with single-cell technologies for enhanced resolution .