ARL8A Antibody

What is ARL8A and what biological functions does it serve?

ARL8A (ADP-ribosylation factor-like protein 8A, also known as ARL10B or GIE2) is a small GTPase superfamily member that plays several critical biological roles. It primarily functions in lysosomal motility and positioning within cells . In neurons specifically, ARL8A mediates the anterograde axonal long-range transport of presynaptic lysosome-related vesicles, which is essential for presynaptic biogenesis and synaptic function . Additionally, research indicates it may play a role in chromosome segregation, although this function is still being characterized . ARL8A has also been identified as a critical component in the efficient phagocytic degradation of apoptotic cells, as demonstrated in studies using C. elegans where ARL-8 was shown to facilitate phagosome-lysosome fusion during phagocytosis .

What are the typical applications for ARL8A antibodies in research?

ARL8A antibodies have been validated for multiple experimental techniques that are commonly used in cellular and molecular research:

These applications allow researchers to detect, quantify, and localize ARL8A protein in various experimental contexts, making them essential tools for studying its expression patterns and functional relationships .

How do I select the appropriate ARL8A antibody for my specific research needs?

When selecting an ARL8A antibody, consider several key factors that will influence experimental success. First, determine your target species—available antibodies have been validated for human, mouse, and rat samples . Next, consider your experimental application; some antibodies are optimized for specific techniques like WB, ICC/IF, IP, or IHC-P. For instance, the rabbit polyclonal antibody (17060-1-AP) has been validated for WB, IHC, IF, IP, and ELISA applications with human and mouse samples .

Specificity is another crucial consideration. Some antibodies target only ARL8A (like EPR24376-2) , while others recognize both ARL8A and ARL8B (like EPR24376-103) . The choice between monoclonal and polyclonal antibodies depends on your specific needs—monoclonals typically offer higher specificity, while polyclonals may provide stronger signals. For critical experiments, consider antibodies that have been validated through knockout/knockdown studies, as mentioned in the product information for some antibodies .

What are the optimal protocols for using ARL8A antibody in Western blot analysis?

For optimal Western blot results with ARL8A antibodies, follow these evidence-based methodological recommendations:

Sample preparation is critical as ARL8A is a small protein (observed at approximately 21-22 kDa) . Use whole cell lysates at appropriate concentrations (typically 40 μg per lane as used in validation studies) . Effective lysis buffers containing protease inhibitors are essential to preserve protein integrity.

For blocking and dilution, use 5% non-fat dry milk in TBST as demonstrated in validation studies . The recommended antibody dilution is 1:1000 for recombinant monoclonal antibodies or 1:500-1:1000 for polyclonal antibodies .

Exposure time should be adjusted based on signal strength—approximately 26 seconds was sufficient for monoclonal antibody detection , while 3 minutes was used for other antibody formats .

ARL8A should be detected at approximately 21-22 kDa . If using antibodies that recognize both ARL8A and ARL8B, be aware that these proteins have similar molecular weights and may appear as a single or closely spaced double band. Validation data shows successful detection in various human cell lines including SH-SY5Y, K-562, SK-BR-3, and HeLa cells, as well as mouse brain tissue .

How should I optimize immunohistochemistry protocols for ARL8A detection in tissue sections?

Successful immunohistochemical detection of ARL8A requires careful optimization of several parameters. Antigen retrieval is particularly important—for paraffin-embedded sections, TE buffer at pH 9.0 is recommended, although citrate buffer at pH 6.0 can serve as an alternative . This step is critical because formalin fixation can mask epitopes necessary for antibody binding.

Antibody concentration should be titrated; a starting dilution of 1:50-1:500 is recommended for polyclonal antibodies in IHC applications . Incubation conditions typically involve overnight incubation at 4°C to achieve optimal signal-to-noise ratio.

Proper controls are essential. Positive control tissues with known ARL8A expression (such as human stomach tissue, which has been validated) should be included alongside experimental samples. Negative controls (omitting primary antibody) help distinguish specific staining from background signal.

For tissue-specific considerations, note that ARL8A expression is particularly relevant in neuronal tissues due to its role in presynaptic vesicle transport . Therefore, special attention to neuronal morphology and subcellular localization patterns is important when interpreting results in brain or nervous system tissues.

What are the recommended procedures for immunofluorescence localization of ARL8A?

For successful immunofluorescence detection of ARL8A, which primarily localizes to lysosomes in mammalian cells , follow these methodological recommendations:

Cell fixation should be performed with 4% paraformaldehyde for 15-20 minutes at room temperature to preserve cellular architecture while maintaining epitope accessibility. For membrane permeabilization, 0.1-0.2% Triton X-100 for 5-10 minutes is typically effective for accessing intracellular ARL8A.

When performing co-localization studies, which are particularly valuable given ARL8A's lysosomal localization, pair the ARL8A antibody with established lysosomal markers (such as LAMP1 or LysoTracker dyes). This approach can provide important functional insights, as demonstrated in C. elegans studies where ARL-8 was shown to affect phagosome-lysosome fusion .

For detection of endogenous versus overexpressed protein, be aware that antibody sensitivity may differ. Endogenous detection may require higher antibody concentrations or more sensitive detection systems. When visualizing results, confocal microscopy is preferable for accurate subcellular localization due to ARL8A's specific localization pattern on lysosomes and its involvement in lysosomal positioning and movement .

How can ARL8A antibodies be used to investigate neurodegenerative disease mechanisms?

ARL8A antibodies serve as valuable tools for investigating neurodegenerative disease mechanisms, particularly in conditions like Parkinson's disease where lysosomal dysfunction is a contributing factor . Research has established links between ARL8A function and neurodegenerative processes, making it an important target for mechanistic studies.

To investigate ARL8A's role in neuronal health and pathology, researchers can employ multiple antibody-based approaches. Immunohistochemistry with ARL8A antibodies can be used to examine expression and localization changes in diseased versus healthy brain tissue. Western blot analysis can quantify potential alterations in ARL8A protein levels in disease models or patient samples. For mechanistic insights, co-immunoprecipitation studies using ARL8A antibodies can identify disease-specific interaction partners that might be disrupted in pathological conditions.

Particularly valuable are studies examining ARL8A's interaction with the HOPS complex component VPS-41, as demonstrated in C. elegans research . This interaction mediates phagosome-lysosome fusion, a process that may be compromised in neurodegenerative conditions. For cellular studies, ARL8A antibodies can be used in neuronal cultures to monitor lysosomal positioning and movement, which is critical for maintaining neuronal health and may be disrupted in disease states .

What approaches can resolve contradictory data when using different ARL8A antibodies?

When faced with contradictory results from different ARL8A antibodies, a systematic troubleshooting approach is essential. First, identify potential sources of variation by comparing antibody characteristics including clone type (monoclonal vs. polyclonal), epitope location, and species reactivity. Some antibodies detect both ARL8A and ARL8B (like EPR24376-103) , while others are specific to ARL8A (like EPR24376-2) , which could explain discrepancies in detection patterns.

Validate antibody specificity through knockout/knockdown controls. Several ARL8A antibodies have been validated in KD/KO systems according to published research . These controls can definitively determine whether observed signals are specific to ARL8A.

Cross-validate findings using multiple detection methods. If Western blot and immunofluorescence results differ, this might indicate epitope accessibility issues in different sample preparation methods. Additionally, employ orthogonal approaches that don't rely on antibodies, such as RNA-level validation through qPCR or RNA-seq to confirm expression patterns.

Consider isoform-specific detection issues. ARL8A and ARL8B share significant homology, and some cellular functions may involve both proteins. Careful selection of isoform-specific antibodies or complementary genetic approaches (such as isoform-specific knockdown) can help resolve whether observed effects are attributable to ARL8A specifically.

How can ARL8A antibodies be employed in lysosomal dysfunction research?

ARL8A antibodies are instrumental in researching lysosomal dysfunction due to ARL8A's critical role in lysosomal positioning and movement . For studying lysosomal trafficking defects, immunofluorescence with ARL8A antibodies can visualize changes in lysosomal distribution patterns in disease models or after experimental manipulations.

To investigate phagosome-lysosome fusion defects, researchers can utilize ARL8A antibodies in co-localization studies with markers for phagosomes and lysosomes. This approach has been effectively demonstrated in C. elegans research, where ARL-8 was shown to facilitate apoptotic cell removal by mediating phagolysosome formation . In those studies, ARL-8 mutants exhibited defects in phagosome-lysosome fusion, leading to the accumulation of RAB-7-positive phagosomes and delayed degradation of apoptotic cells.

For examining protein-protein interactions, co-immunoprecipitation using ARL8A antibodies can identify binding partners involved in lysosomal function. ARL8A physically interacts with the HOPS complex component VPS-41 , and alterations in these interactions may underlie certain lysosomal pathologies.

When investigating lysosomal acidification, which can be assessed using LysoTracker or acridine orange staining as demonstrated in C. elegans studies , ARL8A antibodies can provide complementary information about lysosomal protein machinery that might be affected in conditions with impaired acidification.

What are the latest findings regarding ARL8A's role in cellular processes beyond lysosomal functions?

Recent research has expanded our understanding of ARL8A's cellular roles beyond its established lysosomal functions. While ARL8A's involvement in lysosomal positioning and movement is well-documented , emerging evidence suggests roles in additional cellular processes.

Studies in C. elegans have revealed ARL-8's crucial function in apoptotic cell removal through mediating phagosome-lysosome fusion . In this context, ARL-8 physically interacts with the homotypic fusion and protein sorting (HOPS) complex component VPS-41 to facilitate fusion events between phagosomes and lysosomes. This finding opens new avenues for investigating ARL8A's role in cellular clearance mechanisms that may be relevant to various physiological and pathological conditions.

Additionally, there are indications that ARL8A may play a role in chromosome segregation , although this function requires further characterization. This potential involvement in mitotic processes would represent a significant expansion of ARL8A's known cellular functions and could have implications for understanding its role in cell division and possibly cancer biology.

In neurons specifically, ARL8A mediates anterograde axonal long-range transport of presynaptic lysosome-related vesicles that are required for presynaptic biogenesis and synaptic function . This specialized role highlights ARL8A's tissue-specific functions and suggests it may have additional undiscovered roles in other specialized cell types.

How can ARL8A antibodies be integrated into multi-parameter imaging approaches?

Integrating ARL8A antibodies into multi-parameter imaging approaches enables comprehensive analysis of lysosomal dynamics and interactions with other cellular components. For multiplex immunofluorescence studies, researchers can combine ARL8A antibodies with markers for other organelles to investigate inter-organelle contacts and communication networks. Particularly valuable combinations include ARL8A with markers for endosomes (such as RAB5 and RAB7), autophagosomes, mitochondria, or the endoplasmic reticulum.

For live-cell imaging applications, while antibodies themselves aren't suitable for live imaging, findings from fixed-cell ARL8A antibody studies can inform the design of live-cell experiments using fluorescently tagged ARL8A constructs. This complementary approach can validate dynamic processes observed in fixed specimens using antibodies.

Super-resolution microscopy techniques enhance the utility of ARL8A antibodies by resolving subcellular structures below the diffraction limit. These approaches are particularly valuable for studying lysosomal positioning, which is a key function of ARL8A . Techniques such as STORM, PALM, or SIM can reveal precise spatial relationships between ARL8A-positive structures and other cellular components.

For correlative light and electron microscopy (CLEM), ARL8A antibodies can first identify regions of interest by fluorescence microscopy, which can then be examined at ultrastructural resolution using electron microscopy. This approach is especially useful for examining fine details of lysosome-phagosome fusion events, which have been shown to involve ARL8A in C. elegans studies .

What considerations should be taken when using ARL8A antibodies in protein-protein interaction studies?

When employing ARL8A antibodies for protein-protein interaction studies, several methodological considerations are essential for robust results. For co-immunoprecipitation experiments, antibody selection is critical—choose antibodies that have been validated specifically for immunoprecipitation applications, such as the polyclonal antibody that has been tested for IP in mouse brain tissue .

Given ARL8A's small size (21-22 kDa) , tag interference is an important consideration. If using tagged versions of ARL8A for pulldown assays, ensure that tags do not interfere with protein-protein interactions. Validation with untagged proteins using direct ARL8A antibodies can confirm that observed interactions are not artifacts of tagging.

Nucleotide binding state can significantly affect interaction profiles of small GTPases like ARL8A. Consider performing interaction studies under conditions that stabilize specific nucleotide-bound states (GDP vs. GTP) to capture state-specific interactions. This approach may reveal condition-specific binding partners that are missed in standard assays.

Previous research has identified biologically significant interactions between ARL-8 and the HOPS complex component VPS-41 in C. elegans . This provides a valuable positive control for interaction studies and suggests that examining HOPS complex components as interaction partners in mammalian systems may be particularly fruitful. Additionally, given ARL8A's role in lysosomal positioning and movement , interactions with microtubule motors or their adaptors represent another promising avenue for investigation.

How can I address weak or inconsistent signals when using ARL8A antibodies in Western blot?

When encountering weak or inconsistent signals with ARL8A antibodies in Western blot applications, several optimization strategies can improve results. For sample preparation issues, ensure complete protein extraction using appropriate lysis buffers—RIPA buffer supplemented with protease inhibitors is generally effective for ARL8A extraction. Additionally, since ARL8A is a small protein (21-22 kDa) , use gradient gels (4-20%) to improve resolution in this molecular weight range.

If antibody sensitivity is a concern, implementing signal amplification systems such as enhanced chemiluminescence (ECL) can help. The validated Western blot protocols for ARL8A detection used exposure times ranging from 26 seconds to 3 minutes , suggesting that longer exposure times may be needed depending on the antibody and detection system.

Protein loading should be optimized—validation studies for ARL8A antibodies typically used 40 μg of total protein per lane , which provides a starting point for optimization. Transfer efficiency for small proteins like ARL8A can be improved by using PVDF membranes with smaller pore sizes (0.2 μm rather than 0.45 μm) and adjusting transfer conditions to prevent small proteins from passing through the membrane.

If issues persist, consider tissue/cell type variability—ARL8A expression levels may differ between tissues and cell types. Western blot validation data shows successful detection in various human cell lines (SH-SY5Y, K-562, SK-BR-3, HeLa) and mouse brain tissue , which can guide sample selection.

What strategies can resolve non-specific binding or background issues in immunofluorescence?

Non-specific binding and high background in immunofluorescence can obscure genuine ARL8A signals, particularly given its specific localization to lysosomes in mammalian cells . Several strategies can address these challenges. Optimizing blocking conditions is essential—try different blocking agents (BSA, normal serum, commercial blocking buffers) and increase blocking time (1-2 hours at room temperature or overnight at 4°C) to reduce non-specific binding.

Antibody concentration should be carefully titrated. While specific dilution recommendations may vary by manufacturer, establishing a dilution curve can help identify the optimal concentration that maximizes specific signal while minimizing background. Additional washing steps (increasing wash duration or number of washes) with 0.1% Tween-20 in PBS can help remove unbound antibody.

Pre-adsorption of antibodies can be effective for particularly problematic backgrounds. Incubate the diluted antibody with cell/tissue lysate from a species different from your experimental sample to remove antibodies that may cross-react with non-specific epitopes.

For autofluorescence issues, which can be particularly problematic in certain tissues, treatments such as Sudan Black B (0.1-0.3% in 70% ethanol) can reduce autofluorescence from lipofuscin or other endogenous fluorescent compounds. Alternatively, confocal microscopy with narrow bandpass filters or spectral unmixing can help distinguish true signal from autofluorescence.

How do I interpret and validate unusual localization patterns observed with ARL8A antibodies?

When unusual ARL8A localization patterns are observed, systematic validation approaches are necessary to determine whether they represent genuine biological findings or technical artifacts. First, confirm findings using multiple antibodies targeting different epitopes of ARL8A. If available, both monoclonal (like EPR24376-2) and polyclonal antibodies should be used to verify the observed localization pattern.

Implement genetic validation through siRNA/shRNA knockdown or CRISPR knockout of ARL8A, followed by immunostaining with the same antibody. Disappearance of the unusual localization pattern in knockdown/knockout cells would confirm specificity. Conversely, overexpression of tagged ARL8A can be used to see if the overexpressed protein shows the same unusual localization.

The established lysosomal localization of ARL8A provides a reference point for evaluation. Co-localization studies with established organelle markers (like LAMP1 for lysosomes) can determine whether the unusual pattern represents relocalization of ARL8A or non-specific antibody binding. In C. elegans studies, ARL-8 localization to lysosomes was crucial for its function in phagosome-lysosome fusion , suggesting that any non-lysosomal localization should be carefully validated.

Consider physiological conditions that might alter ARL8A localization. For instance, cellular stress, cell cycle stage, or specific signaling events might cause redistribution of ARL8A from its typical lysosomal location. Performing time-course experiments or stimulating cells with various agents could reveal whether the unusual pattern represents a dynamic response to cellular conditions.