DNAL4 Antibody

What is DNAL4 and why is it significant for research?

DNAL4 (Dynein Axonemal Light Chain 4) is a 12 kDa protein component of the dynein complex. It has gained significant research attention due to its implication in congenital mirror movement disorder (MRMV) and potential role in axonemal-based dynein complexes . DNAL4 is believed to play a role in the cytoplasmic dynein complex for netrin-1-directed retrograde transport, particularly in commissural neurons of the corpus callosum, which may explain its involvement in brain wiring and mirror movements .

What types of DNAL4 antibodies are available for research purposes?

Based on current research tools, DNAL4 antibodies are available in several formats:

Which regions of the DNAL4 protein are typically targeted by research antibodies?

Most commercially available DNAL4 antibodies target either the N-terminal region (amino acids 1-30) or a larger segment (amino acids 1-105). The N-terminal targeting is particularly common for polyclonal antibodies generated in rabbits . This region selection is important as it can affect the antibody's ability to detect truncated or splice variants of DNAL4, including those implicated in genetic disorders .

How should researchers validate DNAL4 antibodies before experimental use?

A systematic approach to antibody validation should include:

• Western blot validation: Verify the antibody detects a band at the expected molecular weight (12 kDa for DNAL4) . Test in tissues known to express DNAL4 such as testis tissue or HEK-293 cells .

• Positive and negative controls: Include tissues/cells with known DNAL4 expression levels. For DNAL4, mouse testis tissue has been validated as a positive control .

• Peptide competition assay: Pre-incubate the antibody with the immunizing peptide before application to verify specificity. This approach has been documented for DNAL4 antibodies in immunohistochemical analysis of human testis tissue .

• Cross-reactivity assessment: Test the antibody against samples from different species if cross-species reactivity is claimed by the manufacturer .

What dilutions are recommended for different applications of DNAL4 antibodies?

What are the optimal sample preparation methods for DNAL4 detection?

For optimal DNAL4 detection across different experimental platforms:

Western Blotting:

• Extract proteins using a lysis buffer containing detergents like NP-40 or Triton X-100

• Include protease inhibitors to prevent degradation

• Denature samples at 95°C for 5 minutes in standard Laemmli buffer

• Use 15% SDS-PAGE gels to better resolve the small (12 kDa) DNAL4 protein

Immunohistochemistry:

• For paraffin-embedded sections, use heat-induced epitope retrieval in citrate buffer (pH 6.0)

• Block with 5-10% normal serum from the same species as the secondary antibody

• Incubate with primary DNAL4 antibody overnight at 4°C at dilutions of 1:50-1:100

Immunoprecipitation:

• Use the appropriate lysis buffer for your cell type

• Optimize antibody and bead amounts

• Include critical controls: input control, isotype control, and bead-only control

• Wash thoroughly to remove non-specifically bound proteins

How can I optimize immunoprecipitation experiments using DNAL4 antibodies?

For effective DNAL4 immunoprecipitation:

• Antibody selection: Choose antibodies specifically validated for IP applications. Note that antibodies validated for native immunoprecipitation may not perform under denaturing conditions .

• Sample preparation: Prepare lysates in non-denaturing buffers that preserve protein-protein interactions if studying DNAL4 complexes.

• Essential controls:

  • Input control: Whole lysate to verify western blot functionality

  • Isotype control: Match the IgG subclass of the primary antibody (e.g., Rabbit IgG for rabbit polyclonal anti-DNAL4)

  • Bead-only control: To identify any non-specific binding to the beads

• Co-IP considerations: When investigating DNAL4 interactions with other proteins in the dynein complex, mild lysis conditions and thorough washing are critical to maintain specific interactions while removing background.

Why might Western blots with DNAL4 antibodies show multiple bands?

Multiple bands in DNAL4 Western blots may occur due to:

• Alternative splice variants: DNAL4 has been documented to undergo alternative splicing, which can produce variant proteins. A notable example is the splice site mutation that causes skipping of exon 3, resulting in a protein missing 28 amino acids .

• Post-translational modifications: DNAL4 may undergo modifications that alter its mobility on SDS-PAGE gels.

• Cross-reactivity: Some antibodies may cross-react with related dynein light chain family members, particularly those with similar molecular weights.

• Degradation products: Improper sample handling or insufficient protease inhibition can result in degradation fragments.

Solution: Validate observed bands using knockout/knockdown controls or peptide competition assays. Consider using multiple antibodies targeting different epitopes of DNAL4 to confirm specificity.

What are common issues with immunohistochemical detection of DNAL4?

Common challenges include:

• Weak signal: DNAL4 is a low abundance protein in many tissues. Enhance detection by:

  • Using signal amplification methods (e.g., biotin-streptavidin systems)

  • Optimizing antigen retrieval methods

  • Increasing antibody concentration or incubation time

  • Using higher sensitivity detection systems

• High background: May result from non-specific binding. Reduce by:

  • Increasing blocking time or concentration

  • Optimizing antibody dilution

  • Using more stringent washing procedures

  • Pre-absorbing the antibody with non-specific proteins

• Tissue-specific considerations: DNAL4 expression varies across tissues. Testis tissue has been validated for DNAL4 antibody testing in immunohistochemistry .

How can DNAL4 antibodies be used to study neurodevelopmental disorders?

DNAL4 mutations have been implicated in congenital mirror movement disorder (MRMV) . Researchers can:

• Compare DNAL4 expression patterns between normal and affected tissues using validated antibodies in immunohistochemistry or immunofluorescence.

• Develop variant-specific antibodies that selectively recognize mutant forms of DNAL4, such as those with exon 3 skipping identified in MRMV patients .

• Use co-immunoprecipitation with DNAL4 antibodies to examine differences in protein interactions between wild-type and mutant DNAL4, potentially revealing disrupted molecular pathways.

• Employ DNAL4 antibodies in brain sections to study the protein's distribution in commissural neurons of the corpus callosum, which may provide insights into the faulty cross-brain wiring observed in MRMV .

What is the relationship between DNAL4 and other genes associated with congenital mirror movements?

DNAL4 is one of five genes (along with DCC, RAD51, NTN1, and ARHGEF7) associated with congenital mirror movements (CMM) . Research using DNAL4 antibodies can explore:

• Co-localization studies: Using multi-label immunofluorescence with antibodies against DNAL4 and other CMM-associated proteins to determine if they operate in the same cellular compartments or pathways.

• Protein interaction networks: Employ co-immunoprecipitation followed by mass spectrometry to identify interactions between DNAL4 and products of other CMM genes.

• Comparative expression analysis: Quantify expression levels of DNAL4 relative to other CMM genes in various neural tissues during development using antibody-based techniques like Western blotting or immunohistochemistry.

These studies may help elucidate whether the various CMM-associated genes operate in common or parallel pathways in neurodevelopment.

What mass spectrometry approaches can be used with DNAL4 immunoprecipitation?

For proteomic analysis of DNAL4 and its interaction partners:

• Sample preparation for LC-MS/MS:

  • After immunoprecipitation, resuspend the bead pellet in denaturing buffer (50 mM Ammonium Bicarbonate with 0.1% RapiGest)

  • Perform reduction (30 mM DTT), alkylation (35 mM Iodoacetamide), and trypsin digestion

  • Acidify to 1% TFA and heat for 30 minutes at 90°C

  • Load 5-15 μl of sample onto the instrument for LC-MS/MS analysis

• Analysis approaches:

  • Bottom-up proteomics to identify post-translational modifications on DNAL4

  • Top-down LC-MS to monitor intact mass and evidence of modifications or truncation

  • Cross-linking mass spectrometry to identify precise interaction interfaces between DNAL4 and binding partners

This combined approach allows for comprehensive characterization of DNAL4 biology in health and disease contexts.

What is the current evidence for DNAL4's role in ciliary function versus cytoplasmic dynein complexes?

Current research presents an interesting dichotomy:

This dual functionality requires further investigation, potentially using DNAL4 antibodies in subcellular localization studies and selective knockdown experiments.

How is DNAL4 research contributing to our understanding of congenital mirror movements?

Research involving DNAL4 has expanded our understanding of congenital mirror movements:

• Genetic heterogeneity: DNAL4 is one of five genes currently associated with CMM, highlighting the genetic complexity of this disorder .

• Novel mechanisms: The discovery of DNAL4 mutations in CMM suggests mechanisms beyond those previously identified with DCC and RAD51 mutations, potentially involving dynein-mediated transport processes .

• Recessive inheritance: While DCC-related CMM typically shows autosomal dominant inheritance, DNAL4 mutations were identified in a family with apparent autosomal recessive inheritance, broadening our understanding of CMM genetics .

• Corpus callosum development: DNAL4's presumed role in netrin-1-directed transport in commissural neurons suggests its importance in the formation of brain commissures, providing insight into the neuroanatomical basis of mirror movements .

What are emerging applications for DNAL4 antibodies in developmental neuroscience?

Emerging applications include:

• Developmental expression mapping: Using DNAL4 antibodies to track expression during critical periods of commissural axon guidance and corpus callosum formation.

• 3D brain organoid studies: Applying DNAL4 immunofluorescence to human brain organoids to observe its role in human-specific neurodevelopmental processes.

• Super-resolution microscopy: Employing DNAL4 antibodies with techniques like STORM or PALM to visualize the precise subcellular localization of DNAL4 in growth cones and axonal transport mechanisms.

• In vivo imaging: Developing fluorescently-tagged antibody fragments for live imaging of DNAL4 dynamics in developing neural circuits.

These approaches may provide unprecedented insights into DNAL4's function in neurodevelopment and potentially reveal new therapeutic targets for associated disorders.