MAP1S Antibody

What is MAP1S and what cellular functions should researchers consider when studying it?

MAP1S (microtubule-associated protein 1S) is a ubiquitously distributed homologue of the neuronal-specific microtubule-associated proteins MAP1A and MAP1B. It generates multiple isoforms through post-translational modification similar to other MAP1 family members. While the calculated molecular weight is 85 kDa (806 amino acids), it typically appears at 130-150 kDa in experimental detection systems .

MAP1S serves as a crucial bridge between microtubules and autophagy mechanisms. It interacts with autophagosome-associated light chain 3 (LC3) and recruits it to stable microtubules in an isoform-dependent manner . Additionally, MAP1S interacts with mitochondrion-associated LRPPRC (leucine-rich PPR-motif containing protein), suggesting involvement in mitophagy. Research designs should account for these multilayered functions when investigating MAP1S roles in cellular processes .

What are the validated applications for MAP1S antibody detection systems?

The MAP1S antibody (15695-1-AP) has been extensively validated across multiple applications with specific detection patterns:

The antibody has been cited in at least 5 publications for WB, 3 for IF, and 1 for IP applications, demonstrating its reliability across multiple experimental settings . When designing experiments, researchers should consider that optimal dilutions may be sample-dependent and should be determined empirically for each experimental system.

How do MAP1S antibodies distinguish between different protein isoforms?

MAP1S gives rise to multiple isoforms through post-translational modifications. The 15695-1-AP antibody was generated against a specific immunogen sequence (amino acids 457-806 encoded by BC008806) , enabling it to recognize distinct MAP1S isoforms. Western blot analysis in human brain tissue typically detects two main bands between 100-130 kDa, corresponding to the heavy and light chains of MAP1S .

When interpreting results, researchers should note that MAP1S isoforms interact differently with LC3 and microtubules. The full-length (FL), heavy chain (HC), and short chain (SC) isoforms of MAP1S show differential binding affinities and functional properties . Experimental designs must account for these isoform-specific interactions when investigating MAP1S roles in autophagy and microtubule dynamics.

What are the optimal sample preparation methods for detecting MAP1S in different cellular compartments?

For successful detection of MAP1S across cellular compartments, sample preparation should be tailored to preserve both protein integrity and spatial organization:

For western blotting, researchers should use lysis buffers containing protease inhibitors to prevent degradation of MAP1S, which is particularly important given its susceptibility to post-translational processing. The differential appearance of MAP1S in Western blots (observed at 130-150 kDa versus calculated 85 kDa) indicates potential glycosylation or other modifications that require careful sample handling .

For immunohistochemistry applications, antigen retrieval is critical. The recommended protocol suggests using TE buffer at pH 9.0, although citrate buffer at pH 6.0 can serve as an alternative . This step is essential for exposing MAP1S epitopes that may be masked during fixation procedures, particularly in tissues like prostate and pancreatic cancer samples where positive detection has been validated.

For immunofluorescence studies investigating the colocalization of MAP1S with autophagy markers or microtubules, fixation with 4% paraformaldehyde followed by permeabilization with 0.1% Triton X-100 has proven effective in preserving the structural relationships between MAP1S and its binding partners .

How should researchers design MAP1S knockout validation experiments?

MAP1S knockout validation is essential for confirming antibody specificity and for studying MAP1S functions. Based on published approaches, researchers should consider the following methodology:

The most effective MAP1S knockout strategy targets exons 4-7, which represent 88.9% of the coding region and cover all putative functional domains, including the microtubule-binding domain, MAGD (mitochondria aggregation and genomic destruction) domain, and actin-binding domain . This approach ensures complete functional ablation of MAP1S.

For experimental validation of the knockout, researchers should employ both PCR-based genotyping and protein-level confirmation through Western blotting. When using the 15695-1-AP antibody for knockout confirmation, the complete absence of both heavy and light chain bands (100-130 kDa) should be observed in the knockout samples compared to wild-type controls .

To study specific MAP1S functions in autophagy, researchers can generate hybrid models such as GFP-LC3 × Map1s−/− mice, which allow visualization of autophagy processes in the absence of MAP1S . This approach is particularly valuable for investigating MAP1S roles in autophagosome formation and trafficking.

What controls are essential when using MAP1S antibodies in immunofluorescence studies?

For rigorous immunofluorescence studies involving MAP1S antibodies, the following controls are indispensable:

Primary antibody specificity control: Include MAP1S knockout or knockdown samples alongside wild-type samples. The 15695-1-AP antibody has been validated in IF/ICC applications with U2OS and HeLa cells at dilutions of 1:200-1:800 .

Positive colocalization controls: When studying MAP1S interactions with microtubules, include α-tubulin co-staining. For autophagy studies, LC3 co-staining is essential. These approaches help visualize the bridging function of MAP1S between microtubules and autophagy machinery .

Negative controls: Include samples without primary antibody and secondary-only controls to assess non-specific binding of secondary antibodies. Additionally, samples treated with microtubule-disrupting agents (e.g., nocodazole) can serve as functional negative controls for MAP1S-microtubule interactions .

Treatment validation controls: For studies investigating MAP1S roles in autophagy, include rapamycin-treated samples (autophagy inducer) and bafilomycin A1-treated samples (autophagy flux inhibitor) to validate autophagy-dependent responses .

How can researchers effectively study the role of MAP1S in autophagy mechanisms?

Investigating MAP1S functions in autophagy requires sophisticated experimental approaches that capture its dual interactions with microtubules and autophagic machinery:

For protein interaction studies, researchers should employ co-immunoprecipitation assays using the MAP1S antibody (0.5-4.0 µg for 1.0-3.0 mg of total protein) to pull down MAP1S complexes, followed by immunoblotting for autophagy proteins such as LC3 . Alternatively, researchers can express tagged MAP1S constructs (HA-MAP1SFL, HA-MAP1SHC, HA-MAP1SSC) to study isoform-specific interactions with autophagy components .

For dynamic visualization of MAP1S-autophagy interactions, dual-color live cell imaging with fluorescently tagged MAP1S and LC3 provides insights into the temporal aspects of their association. This approach has revealed that MAP1S recruits LC3 to stable microtubules in an isoform-dependent manner .

For functional studies, comparative analysis of autophagy flux in wild-type versus MAP1S knockout cells provides critical information. Researchers should monitor LC3-I to LC3-II conversion, p62/SQSTM1 degradation, and autophagic vesicle formation using both biochemical and microscopy-based methods. MAP1S knockout models have demonstrated reduced Bcl-2/xL and P27 protein levels and accumulation of defective mitochondria, suggesting impaired autophagosomal biogenesis and clearance .

What methodological approaches reveal MAP1S involvement in phagocytosis and TLR signaling?

MAP1S plays a significant role in phagocytosis and Toll-like receptor (TLR) signaling, which can be investigated through the following methodological approaches:

Phagocytosis assays using fluorescently labeled bacteria (such as GFP-expressing E. coli) should be conducted with wild-type and MAP1S-deficient macrophages. Both confocal microscopy and flow cytometry analyses have confirmed that MAP1S-deleted macrophages exhibit impaired phagocytosis of multiple bacterial strains, including S. aureus, S. typhimurium, and E. coli .

For investigating interactions between MAP1S and TLR signaling components, co-immunoprecipitation assays with the MAP1S antibody followed by immunoblotting for MyD88 (a key TLR adaptor) are effective. Research has demonstrated that MAP1S interacts directly with MyD88 upon TLR activation and affects TLR signaling pathways .

To visualize the role of MAP1S in autophagy-phagocytosis crosstalk, fluorescence colocalization studies should examine MyD88, MAP1S, and LC3 distribution. Intriguingly, upon TLR activation, MyD88 participates in autophagy processing in a MAP1S-dependent manner by co-localizing with LC3 . This indicates a novel connection between phagocytosis and autophagy that involves MAP1S.

How can researchers differentiate between MAP1S and other MAP1 family members in experimental settings?

Distinguishing MAP1S from other MAP1 family members requires careful consideration of multiple factors:

Antibody selection is critical for specificity. The 15695-1-AP antibody targets a specific immunogen sequence (amino acids 457-806) of MAP1S that differs from MAP1A and MAP1B. Researchers should validate antibody specificity through Western blot analysis in tissues known to express different MAP1 family members. While neuronal MAP1A/B are predominantly found in neural tissues, MAP1S is ubiquitously distributed , providing a tissue-based differentiation strategy.

Expression pattern analysis can differentiate MAP1 family members. Unlike the exclusively neuronal distribution of MAP1A/B, MAP1S is expressed in multiple cell types with particularly high expression in macrophages . Cell type-specific analysis can therefore help distinguish MAP1S from other family members.

For functional differentiation, researchers can exploit the unique roles of MAP1S in autophagy and phagocytosis. While all MAP1 family members interact with microtubules, only MAP1S has been demonstrated to bridge autophagic components with microtubules and regulate phagocytosis through TLR signaling . Functional assays focusing on these processes can help separate MAP1S-specific effects from those of other MAP1 family members.

Why might MAP1S antibodies detect multiple bands in Western blots and how should researchers interpret these patterns?

The detection of multiple bands when using MAP1S antibodies is a common observation that requires careful interpretation:

The MAP1S protein undergoes post-translational processing similar to other MAP1 family members, resulting in multiple isoforms. Western blot analysis in human brain tissue typically detects two main bands between 100-130 kDa, representing the heavy and light chains of MAP1S . The calculated molecular weight of MAP1S is 85 kDa (806 amino acids), but the observed molecular weight ranges from 130-150 kDa , indicating extensive post-translational modifications.

Different isoforms of MAP1S (full-length, heavy chain, and short chain) have been documented and serve distinct functions. The full-length form contains all functional domains, while processed forms may interact differently with binding partners such as LC3 . Researchers should consider using isoform-specific constructs (HA-MAP1SFL, HA-MAP1SHC, HA-MAP1SSC) as positive controls to identify specific bands .

To distinguish specific from non-specific bands, researchers should include MAP1S knockout samples as negative controls. Additionally, protein lysates from cells transfected with siRNA targeting MAP1S can help identify which bands decrease in intensity with MAP1S knockdown .

What factors affect MAP1S antibody performance across different experimental applications?

Several factors can influence MAP1S antibody performance, and researchers should consider the following variables:

Sample preparation significantly impacts antibody performance. For Western blotting, complete protein denaturation is crucial, whereas for applications like immunoprecipitation, native protein conformation must be preserved. The 15695-1-AP antibody has been optimized for multiple applications with specific recommendations: 1:1000-1:8000 for WB, 0.5-4.0 μg for IP with 1.0-3.0 mg total protein, and 1:200-1:800 for both IHC and IF/ICC .

Antigen retrieval methods are particularly important for tissue sections. For the 15695-1-AP antibody, TE buffer at pH 9.0 is recommended for antigen retrieval in IHC applications, though citrate buffer at pH 6.0 can serve as an alternative . Insufficient antigen retrieval is a common cause of false-negative results in IHC.

Storage conditions affect antibody stability and performance. The MAP1S antibody should be stored at -20°C in PBS containing 0.02% sodium azide and 50% glycerol (pH 7.3). Under these conditions, it remains stable for one year after shipment, and aliquoting is not necessary for -20°C storage .

Cross-reactivity with other MAP1 family members can occur due to structural similarities. While the 15695-1-AP antibody is designed to be specific for MAP1S, researchers should validate specificity in their experimental systems, particularly when studying tissues or cells that express multiple MAP1 family members .

How can researchers validate MAP1S antibody specificity in their experimental systems?

Validating antibody specificity is essential for generating reliable research data. For MAP1S antibodies, researchers should implement the following validation strategies:

Genetic models provide the gold standard for antibody validation. Researchers should compare antibody reactivity in wild-type versus MAP1S knockout samples. Published studies have generated MAP1S knockout mice by targeting exons 4-7, which represent 88.9% of the coding region and cover all putative functional domains . Complete absence of signal in knockout samples confirms antibody specificity.

RNA interference approaches offer an alternative when knockout models are unavailable. Cells transfected with MAP1S-targeting siRNA or shRNA should show reduced antibody reactivity compared to control transfections. This approach has been successfully used to validate MAP1S antibody specificity in phagocytosis studies .

Recombinant protein expression can provide positive controls. Researchers can express tagged MAP1S constructs (HA-MAP1SFL, HA-MAP1SHC, HA-MAP1SSC) and confirm detection with both the MAP1S antibody and tag-specific antibodies . Concordant detection patterns support antibody specificity.

Peptide competition assays provide additional validation. Pre-incubating the MAP1S antibody with excess immunizing peptide (amino acids 457-806) should eliminate specific signals, while non-specific signals may persist. This approach helps distinguish true MAP1S detection from cross-reactivity.

How can researchers investigate MAP1S roles in disease mechanisms beyond cancer?

MAP1S functions extend beyond cancer to various disease contexts, which can be investigated through the following approaches:

For neurodegenerative diseases, researchers should examine MAP1S interactions with aggregation-prone proteins like tau or α-synuclein. Since MAP1S bridges autophagic components with microtubules and affects autophagic clearance , its dysfunction might contribute to protein aggregation. Immunoprecipitation with MAP1S antibodies followed by mass spectrometry can identify novel disease-relevant interaction partners.

In infectious disease research, MAP1S roles in phagocytosis and TLR signaling merit investigation. MAP1S-deficient macrophages show impaired phagocytosis of multiple bacterial strains . Researchers should explore whether MAP1S polymorphisms or expression levels correlate with susceptibility to bacterial infections through case-control studies combining genotyping and MAP1S immunohistochemistry.

For metabolic disorders, MAP1S involvement in mitochondrial quality control through mitophagy suggests potential roles in diseases characterized by mitochondrial dysfunction. Analysis of MAP1S expression and post-translational modifications in metabolic disease tissues, using the 15695-1-AP antibody at 1:200-1:800 dilution for IHC , could reveal disease-specific alterations.

What advanced imaging techniques can reveal MAP1S dynamics in live cells?

Understanding MAP1S dynamics requires sophisticated imaging approaches that capture its interactions in real-time:

Super-resolution microscopy techniques such as structured illumination microscopy (SIM) or stochastic optical reconstruction microscopy (STORM) can resolve MAP1S interactions with microtubules and autophagic structures beyond the diffraction limit. These approaches require careful sample preparation and potentially direct fluorophore conjugation to the MAP1S antibody.

Fluorescence resonance energy transfer (FRET) between fluorescently tagged MAP1S and its binding partners (such as LC3 or MyD88) can reveal dynamic protein-protein interactions with nanometer resolution. This approach has helped elucidate how MAP1S recruits LC3 to stable microtubules in an isoform-dependent manner .

Correlative light and electron microscopy (CLEM) combines the specificity of fluorescence labeling with the ultrastructural resolution of electron microscopy. This technique is particularly valuable for studying MAP1S localization relative to autophagosomes and mitochondria during mitophagy, providing insights into its role in bridging these structures with microtubules .

For tracking MAP1S mobility along microtubules, fluorescence recovery after photobleaching (FRAP) or photoactivation approaches with fluorescently tagged MAP1S constructs can reveal the dynamic nature of its interactions with the cytoskeleton in response to autophagic stimuli or bacterial challenge.