DNALI1 Antibody

What is DNALI1 and why is it important in cellular research?

DNALI1 belongs to the inner dynein arm light chain family and encodes a subunit of the light intermediate chain of inner dynein arms of the axoneme of sperm flagella . It is expressed in several tissues but shows strongest expression in testis tissue, with dramatic increases during spermiogenesis . The protein is critical for proper sperm flagellum assembly and is implicated in neurodegeneration following traumatic brain injury through autophagosome-lysosome fusion inhibition . Recent research has shown that mutations in DNALI1 can cause male infertility with severe oligoasthenoteratozoospermia in humans, highlighting its significance in reproductive biology .

For optimal effectiveness, DNALI1 antibodies should be stored at -20°C and are typically stable for one year after shipment . The antibodies are usually supplied in a storage buffer containing PBS with 0.02% sodium azide and 50% glycerol at pH 7.3 . Aliquoting is often unnecessary for -20°C storage for smaller volumes (20μl sizes) that contain 0.1% BSA . To maintain antibody integrity, avoid repeated freeze-thaw cycles . If small volumes become entrapped in the seal during shipment and storage, briefly centrifuge the vial on a tabletop centrifuge to dislodge any liquid in the container's cap .

What controls should be included when using DNALI1 antibodies for research?

When designing experiments with DNALI1 antibodies, several controls are essential:

• Positive controls: Use mouse or rat testis tissue for Western blot validation, as these have been consistently shown to express DNALI1 .

• Negative controls: Include samples where DNALI1 is known to be absent or knockdown/knockout samples using DNALI1 CRISPR/Cas9 KO plasmids .

• Antibody validation controls:

  • For IP experiments: Include no-antibody controls and isotype-matched irrelevant antibody controls

  • For Co-IP: As demonstrated in published protocols, conduct control experiments using COS-1 cells co-transfected with plasmids expressing DNALI1/FLAG and unrelated proteins .

• Cross-reactivity assessment: Test reactivity in multiple species (human, mouse, rat) as documented in published applications .

How can researchers optimize Western blot protocols for DNALI1 detection?

For optimal Western blot detection of DNALI1:

• Sample preparation: Use fresh testis tissue or cells expressing DNALI1, with appropriate protease inhibitors to prevent degradation.

• Protein loading: Load 20-50 μg of total protein per lane, as DNALI1 is detected at approximately 30-33 kDa .

• Antibody dilution: Start with a 1:2000 dilution and optimize based on signal-to-noise ratio . Published protocols recommend dilutions ranging from 1:500 to 1:10000 depending on the specific antibody and sample .

• Detection optimization:

  • Use chemiluminescent detection systems for highest sensitivity

  • For weak signals, consider using signal enhancement systems or longer exposure times

  • When detecting multiple proteins, the observed molecular weight (30-33 kDa) can be used to distinguish DNALI1 from other proteins .

• Blocking optimization: Use 5% non-fat dry milk or BSA in TBST for blocking, with overnight primary antibody incubation at 4°C for best results.

What are the recommended protocols for immunofluorescence detection of DNALI1?

Based on successful published immunofluorescence studies with DNALI1:

• Sample preparation:

  • For tissue sections: Formalin-fixed, paraffin-embedded tissues with heat-mediated antigen retrieval using sodium citrate buffer (pH 6.0) .

  • For cells: Fix with 4% paraformaldehyde for 15 minutes at room temperature.

• Co-localization studies:

  • DNALI1 co-localizes with α-tubulin (a manchette marker) in elongating spermatids .

  • Double staining with anti-DNALI1 polyclonal antibody and anti-PACRG monoclonal antibody has been successful for co-localization studies .

• Imaging recommendations:

  • Use confocal microscopy for high-resolution co-localization studies (e.g., Zeiss LSM 700) .

  • DNALI1 appears closer to the nuclear membrane while PACRG is on the surface of DNALI1 in elongating spermatids .

• Signal amplification: For weak signals, consider using tyramide signal amplification or higher antibody concentrations.

How can researchers investigate DNALI1 interactions with the MEIG1/PACRG complex?

To investigate DNALI1 interactions with the MEIG1/PACRG complex, researchers can employ multiple complementary approaches:

• Co-immunoprecipitation:

  • Use anti-PACRG antibody to pull down complexes from mouse testis extracts

  • Perform western blot with anti-DNALI1 antibody to detect interaction

  • Control experiments should include immunoprecipitation with isotype-matched control antibodies

• Recombinant protein expression and purification:

  • Generate expression constructs for DNALI1/Flag and PACRG/pEGFP-N2 using the following primers:

    • Pacrg forward: 5'-GAATTCATGCCGAAGAGGACTAAACTG-3'

    • Pacrg reverse: 5'-GGATCCGGTTCAGCAAGCACGACTC-3'

    • Dnali1 forward: 5'-GAATTCAATGATACCCCCAGCAGACTCTCTG-3'

    • Dnali1 reverse: 5'-CTCGAGTCACTTCTTCGGTGCGATAATGCC-3'

• Co-localization studies in cellular models:

  • Transfect CHO cells with PACRG/pEGFP-N2 and DNALI1/Flag constructs individually or together

  • Process cells 48 hours post-transfection for immunofluorescence with anti-Flag antibody

  • Analyze using confocal microscopy

• Protein stabilization analysis:

  • DNALI1 stabilizes PACRG in mammalian cells and bacteria, with co-expression dramatically increasing PACRG protein levels

  • This functional association provides additional evidence for their interaction

What techniques can be used to study DNALI1's role in sperm flagellum assembly?

To investigate DNALI1's role in sperm flagellum assembly, researchers can employ these advanced techniques:

• CRISPR/Cas9 knockout models:

  • Use DNALI1 CRISPR/Cas9 KO plasmids (available for human and mouse models)

  • Analyze the resulting phenotype for defects in flagellum structure and function

  • Compare with HDR plasmids for gene replacement studies

• Genetic association studies:

  • Screen for DNALI1 mutations in patients with male infertility or primary ciliary dyskinesia

  • Correlate specific mutations with phenotypic presentations

  • Recent research has linked DNALI1 mutations to male infertility with severe oligoasthenoteratozoospermia

• High-resolution microscopy of flagellar structures:

  • Use electron microscopy to analyze axonemal structure in DNALI1-deficient models

  • Apply super-resolution microscopy techniques to visualize protein localization in intact flagella

  • Compare wild-type and mutant structures to identify specific defects

• Functional motility assays:

  • Analyze sperm motility parameters using computer-assisted sperm analysis (CASA)

  • Correlate DNALI1 expression levels with motility parameters

  • Test rescue of motility defects with wild-type DNALI1 expression

How can researchers investigate DNALI1's role in autophagy inhibition following traumatic brain injury?

Recent research has shown that DNALI1 promotes neurodegeneration after traumatic brain injury via inhibition of autophagosome-lysosome fusion . To further investigate this role:

• In vitro models of neuronal injury:

  • Establish primary neuronal cultures or neuronal cell lines with modulated DNALI1 expression

  • Apply oxygen-glucose deprivation or mechanical injury models

  • Analyze autophagosome formation and lysosomal fusion using fluorescent markers and co-localization studies

• Autophagy flux analysis:

  • Use tandem fluorescent-tagged LC3 (mRFP-GFP-LC3) to distinguish autophagosomes from autolysosomes

  • Quantify the effect of DNALI1 knockdown or overexpression on autophagy flux

  • Apply autophagy inhibitors and activators to determine the stage of autophagy affected by DNALI1

• Protein-protein interaction studies:

  • Identify potential DNALI1 binding partners in the autophagy machinery using co-immunoprecipitation followed by mass spectrometry

  • Validate interactions using proximity ligation assays or FRET-based approaches

  • Map interaction domains through deletion mutant analysis

• In vivo traumatic brain injury models:

  • Compare DNALI1 knockout and wild-type mice in controlled cortical impact or fluid percussion injury models

  • Assess neurodegeneration, autophagy markers, and behavioral outcomes

  • Test therapeutic approaches targeting the DNALI1 pathway

What are common issues when using DNALI1 antibodies in Western blotting and how can they be resolved?

Researchers may encounter several common issues when using DNALI1 antibodies in Western blotting:

• Weak or no signal:

  • Increase antibody concentration (try 1:500 dilution if 1:2000 is insufficient)

  • Extend primary antibody incubation time to overnight at 4°C

  • Increase protein loading (up to 50-75 μg per lane)

  • Check protein transfer efficiency with Ponceau S staining

  • Verify sample integrity and protein expression in positive control tissues (mouse testis)

• Multiple bands or non-specific binding:

  • Increase blocking time and concentration (5-10% blocking agent)

  • Use more stringent washing conditions (higher salt concentration or 0.1% SDS in wash buffer)

  • Lower primary antibody concentration (1:10000 dilution)

  • Confirm specificity using DNALI1 knockout samples as negative controls

  • The expected molecular weight for DNALI1 is 30-33 kDa

• High background:

  • Increase washing duration and frequency

  • Decrease primary and secondary antibody concentrations

  • Use fresher antibody aliquots to avoid degradation

  • Consider different blocking agents (milk vs. BSA)

• Inconsistent results between experiments:

  • Standardize protein extraction methods

  • Use the same positive controls across experiments

  • Maintain consistent antibody handling and storage conditions

  • Document lot-to-lot variations in antibody performance

How can researchers validate the specificity of DNALI1 antibodies?

Thorough validation of DNALI1 antibody specificity is critical for reliable research findings:

• Knockout/knockdown controls:

  • Use CRISPR/Cas9 knockout cells or tissues

  • Apply siRNA or shRNA knockdown of DNALI1

  • Compare signal between wild-type and DNALI1-deficient samples across applications

• Peptide competition assays:

  • Pre-incubate antibody with excess immunizing peptide

  • Compare signal with and without peptide competition

  • Specific signals should be significantly reduced or eliminated

• Cross-validation with multiple antibodies:

  • Test antibodies from different sources targeting distinct epitopes

  • Compare staining patterns across applications

  • Multiple antibodies with convergent results increase confidence in specificity

• Correlation with mRNA expression:

  • Compare protein detection with RT-PCR or RNA-seq data

  • Tissues with high mRNA expression (e.g., testis) should show corresponding protein levels

  • DNALI1 mRNA expression dramatically increases during spermiogenesis

• Mass spectrometry validation:

  • Immunoprecipitate DNALI1 and confirm identity by mass spectrometry

  • This approach provides the highest level of validation for antibody specificity

What considerations should researchers keep in mind when selecting a DNALI1 antibody for specific applications?

When selecting DNALI1 antibodies for specific applications, researchers should consider:

• Epitope location and antibody type:

  • Antibodies targeting different regions (N-terminus, C-terminus, or internal domains) may perform differently in various applications

  • Available options include antibodies targeting:

    • Full-length protein (AA 1-258)

    • N-terminal region

    • C-terminal region

    • Specific domains (e.g., AA 201-250)

• Host species and clonality:

  • Polyclonal rabbit antibodies are commonly used and show broad reactivity

  • Monoclonal mouse antibodies (e.g., clone 2H3) provide consistent lot-to-lot performance

  • Consider host species when designing multi-color immunofluorescence experiments

• Validated applications:

  • Some antibodies are validated for multiple applications (WB, IF, IP, ELISA)

  • Others may be optimized for specific applications

  • Review application-specific validation data before selection

• Species reactivity:

  • Confirm reactivity with species of interest (human, mouse, rat)

  • Some antibodies show broader cross-reactivity with additional species (cow, dog, guinea pig, horse, rabbit, zebrafish, bat, pig)

  • Test new species applications with appropriate positive controls

• Research context:

  • For co-localization studies with PACRG, select antibodies used in published studies

  • For developmental studies, use antibodies validated in testis tissue during spermatogenesis

  • For neurodegeneration research, confirm antibody performance in neural tissues

What emerging techniques might enhance DNALI1 research beyond traditional antibody applications?

Several emerging techniques offer potential for advancing DNALI1 research:

• CRISPR-based tagging:

  • Endogenous tagging of DNALI1 with fluorescent proteins or epitope tags

  • Alleviates reliance on antibodies and allows live-cell imaging

  • Enables precise tracking of DNALI1 dynamics during flagellar assembly

• Proximity labeling approaches:

  • APEX2 or BioID fusion to DNALI1 to identify proximal proteins in situ

  • Provides comprehensive identification of the DNALI1 interactome

  • Can reveal previously unknown interaction partners in specific cellular compartments

• Super-resolution microscopy:

  • STORM, PALM, or STED microscopy for nanoscale localization

  • Precise mapping of DNALI1 within axonemal structures

  • Enhanced resolution of co-localization with interaction partners

• Single-molecule tracking:

  • Study dynamics of individual DNALI1 molecules during flagellar assembly

  • Measure binding kinetics and residence times in living cells

  • Correlate molecular behavior with flagellar function

• Cryo-electron tomography:

  • High-resolution structural analysis of DNALI1 within the axonemal complex

  • Determine precise molecular architecture of dynein arms

  • Compare structures in wild-type and disease models

How might researchers develop therapeutic approaches targeting DNALI1 for related diseases?

For developing therapeutic approaches targeting DNALI1-related diseases:

• Gene therapy strategies:

  • AAV-mediated delivery of functional DNALI1 to affected tissues

  • CRISPR-based correction of disease-causing mutations

  • Evaluate delivery efficiency and functional rescue in animal models

• Small molecule screening:

  • Identify compounds that stabilize mutant DNALI1 proteins

  • Develop molecules that enhance remaining DNALI1 function

  • Target downstream pathways affected by DNALI1 dysfunction

• Protein replacement approaches:

  • Develop cell-penetrating DNALI1 protein variants

  • Evaluate localization and functional rescue

  • Optimize delivery to relevant tissues (respiratory epithelium, testis)

• In vitro disease modeling:

  • Patient-derived iPSCs differentiated into relevant cell types

  • Organoid models of ciliated tissues

  • High-throughput screening platforms for drug discovery

• Clinical biomarker development:

  • DNALI1 expression profiling in accessible specimens

  • Correlation with disease severity and progression

  • Monitoring therapeutic response in clinical trials

What are the most promising directions for understanding DNALI1's role beyond reproductive biology?

While DNALI1's role in sperm flagella is well-established, emerging research suggests broader functions:

• Neuronal function and neurodegeneration:

  • Further investigate DNALI1's role in autophagy inhibition after traumatic brain injury

  • Explore potential involvement in other neurodegenerative conditions

  • Examine DNALI1 expression and function in neuronal cilia

• Primary ciliary dyskinesia:

  • Defects in DNALI1 are associated with primary ciliary dyskinesia type 16 (CILD16)

  • Develop more precise genotype-phenotype correlations

  • Evaluate respiratory phenotypes in DNALI1-deficient models

• Cancer biology:

  • Investigate potential roles in cancer cell migration or invasion

  • Examine expression in various tumor types

  • Evaluate prognostic significance in cancer datasets

• Developmental biology:

  • Study DNALI1's role in embryonic node cilia and left-right asymmetry

  • Investigate potential functions in other motile cilia during development

  • Examine conservation of function across evolutionary diverse species

• Immune function:

  • Explore DNALI1's role in ciliated immune cells

  • Investigate potential involvement in mucociliary clearance

  • Study implications for respiratory infections and inflammation