MAB21L1 Antibody

What is MAB21L1 and what cellular processes is it involved in?

MAB21L1 functions as a nuclear factor that modulates both lens-specific gene expression and DNA/nucleotide metabolic processes during lens placode formation. This protein plays a crucial role in ocular development, with mutations causing congenital cerebellar, ocular, craniofacial, and genital (COFG) syndrome . Research using single-cell RNA sequencing has identified 21 lens-specific genes regulated by MAB21L1, including three key genes involved in lens formation: Pitx3, Maf, and Sfrp2. Gene ontology analysis also revealed MAB21L1's involvement in regulating housekeeping genes associated with DNA/nucleotide metabolism prior to cell death during lens development .

What experimental models are available to study MAB21L1 function?

Several experimental models have been developed to investigate MAB21L1 function:

• Mab21l1-null mice: These display severe cell-autonomous defects in lens formation leading to microphthalmia, serving as a model for COFG syndrome .

• CRISPR-Cas9 generated mouse models: Specific mutations like the Mab21l1 p.Arg51Leu substitution have been created in C57BL/6JCrl mice to study the effects of clinically relevant variants .

• Inducible human cell lines: HEK293 Flp-In T-REx system expressing wild-type or mutant forms of MAB21L1 allow for controlled investigation of protein function in a human cellular context .

• Embryonic tissue studies: Wild-type and mutant embryos at specific developmental stages (e.g., E9.5, 24- to 26-somite stages) provide insights into MAB21L1's role during development .

What types of MAB21L1 antibodies are commercially available?

Commercial MAB21L1 antibodies include rabbit polyclonal antibodies such as those offered by Atlas Antibodies . These antibodies are manufactured using standardized processes to ensure quality and reproducibility, and are typically validated for multiple applications including immunohistochemistry (IHC), immunocytochemistry-immunofluorescence (ICC-IF), and Western blotting (WB) . When selecting an antibody, researchers should consider the specific application, host species, and validation data provided by the manufacturer.

What validation methods are typically used for MAB21L1 antibodies?

Rigorous validation ensures antibody specificity and reliability. For MAB21L1 antibodies, common validation methods include:

• Immunohistochemistry (IHC): Testing on tissues known to express MAB21L1, such as developing eye structures .

• Western blotting: Confirming specific detection at the expected molecular weight.

• Genetic validation: Using knockout tissues as negative controls, as demonstrated in studies where MAB21L1 signals were absent in Mab21l1-null embryos .

• Cross-species reactivity assessment: Evaluating antibody performance across different model organisms.

• Batch-to-batch consistency testing: Ensuring reproducible performance across manufacturing lots.

How can I optimize immunohistochemical staining using MAB21L1 antibodies?

Successful immunohistochemical detection of MAB21L1 requires careful optimization:

• Sample preparation: For developmental studies, precise staging is critical (e.g., embryonic day 9.5 or 24-26 somite stage for mouse embryos) .

• Fixation: Use appropriate fixatives like Davidson's fixative for eye tissues to preserve morphology while maintaining antigen accessibility .

• Antibody dilution: Titrate antibody concentration to achieve optimal signal-to-noise ratio.

• Detection system: For nuclear proteins like MAB21L1, choose detection systems with high nuclear penetration efficiency.

• Controls: Include both positive controls (wild-type tissue) and negative controls (Mab21l1-null tissue) .

• Counterstaining: Nuclear counterstains help confirm the expected nuclear localization of MAB21L1.

• Signal analysis: Quantify signal intensity in relation to nuclei to assess expression levels accurately.

What are known protein interaction partners of MAB21L1 and how can they be detected?

Immunoprecipitation followed by mass spectrometry (IP-MS) has identified several MAB21L1 interaction partners. The table below summarizes key interactions:

For detecting these interactions, researchers should:

• Use GFP-tagged or epitope-tagged MAB21L1 for efficient immunoprecipitation

• Perform reciprocal IP with antibodies against suspected interaction partners

• Include appropriate negative controls and biological replicates

• Confirm key interactions through orthogonal methods like co-immunoprecipitation followed by Western blotting

How do mutations in MAB21L1 affect its cellular localization and function?

Mutations in MAB21L1, particularly those affecting the Arg51 residue, have significant functional consequences:

• Subcellular localization: Both wild-type and mutant (Arg51Leu and Arg51Gln) forms of MAB21L1 are present in cytoplasmic and nuclear fractions, suggesting mutations don't significantly alter subcellular distribution .

• Protein interactions: Arg51 mutations alter protein interaction networks. Wild-type MAB21L1 preferentially interacts with TBL1XR1 (NCor co-repressor complex component), while mutant forms show enhanced interaction with RNA-binding proteins like MSI2/Musashi-2 .

• Developmental effects: These mutations cause aniridia and microphthalmia in humans and mouse models . The Mab21l1 p.Arg51Leu mouse model exhibits ocular phenotypes similar to human patients.

• Structural impact: Molecular modeling suggests these mutations affect protein stability and interaction interfaces rather than catalytic activity, as MAB21L1 shows no nucleotidyltransferase activity in enzymatic assays .

What are best practices for using MAB21L1 antibodies in Western blotting?

For optimal Western blot detection of MAB21L1:

• Sample preparation:

  • Consider cellular fractionation to enrich nuclear proteins where MAB21L1 is primarily localized

  • Use appropriate lysis buffers that effectively extract nuclear proteins

  • Include protease inhibitors to prevent degradation

• Electrophoresis conditions:

  • Use gradient gels (4-12% or 4-20%) for optimal resolution

  • Include positive control samples (tissues known to express MAB21L1, such as developing eye tissues)

  • Include negative control samples (Mab21l1-null tissues where available)

• Transfer and detection:

  • Optimize transfer conditions for nuclear proteins (longer transfer times may be needed)

  • Block with appropriate blocking agents to minimize background

  • Use primary antibody concentrations recommended by the manufacturer (typically 0.1-1 μg/ml)

  • Include controls for loading (e.g., GAPDH for cytoplasmic fraction, Histone H3 for nuclear fraction)

• For co-immunoprecipitation experiments:

  • Perform reciprocal IP to confirm interactions (e.g., IP with anti-MAB21L1 and blot for interaction partners, then IP with partner antibody and blot for MAB21L1)

  • Consider using antibodies against both MAB21L1 and its interacting partners for validation

How can MAB21L1 antibodies be used to investigate developmental eye disorders?

MAB21L1 antibodies are valuable tools for investigating ocular developmental disorders:

• Comparative expression analysis: Compare MAB21L1 expression patterns between wild-type and disease models at critical developmental stages. In wild-type embryos, MAB21L1 is expressed in nuclei of lens placode, surface ectoderm, and optic vesicle cells .

• Co-localization studies: Combine MAB21L1 antibodies with markers of ER stress (such as GRP78) to investigate cellular pathology. Studies have shown increased GRP78 signals in the apical cytoplasm of Mab21l1-null lens placode cells, indicating ER stress prior to lens formation defects .

• Developmental phenotyping: Use MAB21L1 antibodies alongside morphological assessment tools like slit lamp biomicroscopy, OCT, and histology to correlate protein expression with developmental anomalies in mouse models .

• Molecular pathology investigations: Combine immunohistochemistry with electron microscopy to examine ultrastructural abnormalities. Mab21l1-null lens placodes show enlarged ER lumens in the apical cytoplasm, correlating with defects in lens formation .

• Therapeutic intervention assessment: Use MAB21L1 antibodies to monitor protein expression/localization following experimental interventions aimed at rescuing developmental defects.

What experimental approaches can be used to study MAB21L1's role in DNA/nucleotide metabolism?

Several methodological approaches can investigate MAB21L1's role in DNA/nucleotide metabolism:

• Transcriptomic analysis: Single-cell RNA sequencing of wild-type versus Mab21l1-null lens ectoderms has identified 279 differentially expressed genes, with significant enrichment in housekeeping genes associated with DNA/nucleotide metabolism .

• Biochemical assays: Although MAB21L1 structurally resembles nucleotidyltransferases, enzymatic assays using purified protein have shown no detectable nucleotidyltransferase activity with ATP, CTP, GTP, or UTP as substrates, with either DNA or RNA as activators . This suggests a non-enzymatic regulatory role.

• Protein interaction studies: IP-MS has identified MAB21L1 interactions with RNA-binding proteins and transcription factors, suggesting it may indirectly regulate nucleotide metabolism through protein-protein interactions .

• Cellular stress markers: Examining markers of ER stress, DNA damage, or replication stress in MAB21L1-deficient cells can provide insights into metabolic consequences of MAB21L1 loss.

• In vivo phenotyping: Correlating metabolic abnormalities with developmental defects in Mab21l1 mutant models can elucidate the functional significance of the protein's role in nucleotide metabolism.