ARFGAP3 Antibody

What applications has the ARFGAP3 antibody been validated for?

The ARFGAP3 antibody (15293-1-AP) has been validated for multiple experimental applications including Western Blot (WB), Immunoprecipitation (IP), Immunohistochemistry (IHC), Immunofluorescence (IF)/Immunocytochemistry (ICC), and ELISA techniques. The antibody has demonstrated specific reactivity in these applications across human, mouse, and rat samples . Published literature confirms successful application in knockout/knockdown validation experiments, with at least one publication demonstrating specificity in KD/KO systems, three publications utilizing the antibody for Western blotting, and one publication employing it for immunofluorescence applications . When designing experiments, researchers should consider that this antibody targets the full ARFGAP3 protein and has been validated with specific cell lines and tissues for each application type.

What are the recommended dilution ratios for different applications?

The optimal dilution varies significantly depending on the experimental application. For accurate results, the following application-specific dilutions are recommended:

It is essential to titrate the antibody in each testing system to obtain optimal results, as experimental conditions and sample types can significantly influence antibody performance . Some applications may require further optimization based on specific sample characteristics.

What positive controls are recommended for ARFGAP3 antibody validation?

For Western blot applications, HepG2 cells, mouse brain tissue, and Jurkat cells have been validated as positive controls that consistently express detectable levels of ARFGAP3 . For immunoprecipitation experiments, HepG2 cells have been confirmed as reliable positive controls . In immunohistochemistry applications, human breast cancer tissue provides a reliable positive control, though researchers should note that antigen retrieval is required (preferably with TE buffer pH 9.0, with citrate buffer pH 6.0 as an alternative) . For immunofluorescence applications, both A431 and HepG2 cells have been confirmed as suitable positive controls . Including these validated controls in experimental designs is crucial for confirming antibody specificity and performance.

How should samples be prepared for optimal ARFGAP3 detection in immunohistochemistry?

For successful ARFGAP3 detection in immunohistochemistry applications, antigen retrieval is essential and significantly impacts results. The recommended protocol involves epitope retrieval with TE buffer at pH 9.0, though citrate buffer at pH 6.0 may be used as an alternative with potentially different sensitivity . After retrieval, the antibody should be used at a dilution of 1:20-1:200, with the optimal concentration determined through experimental titration for each specific tissue type . For immunohistochemical localization studies of ARFGAP3 in neural tissues, particularly retinal sections, thin sectioning combined with proper fixation is critical for preserving the delicate architecture of structures like photoreceptor ribbon synapses . The antibody has been successfully used in super-resolution structured illumination microscopy (SR-SIM), which provides enhanced resolution for precise localization studies in complex neural tissues .

What are the critical considerations for co-immunoprecipitation studies with ARFGAP3?

When conducting co-immunoprecipitation studies to investigate ARFGAP3 protein interactions, several methodological factors are critical. For direct immunoprecipitation of ARFGAP3, use 0.5-4.0 μg of antibody for 1.0-3.0 mg of total protein lysate . Research has demonstrated that ARFGAP3 can be co-immunoprecipitated with RIBEYE from bovine retinal extracts, confirming their interaction in vivo . When designing co-IP experiments, it's important to consider the redox-sensitive nature of certain ARFGAP3 interactions; for example, the ARFGAP3-RIBEYE interaction is NAD(H)-dependent and more efficient with NADH than with oxidized NAD+ . For pull-down experiments involving ARFGAP3's ArfGAP domain, including reducing agents (such as 1mM βME) in the buffer can help maintain proper protein conformation . Control experiments should include pre-immune serum conditions to verify specificity of precipitated complexes, as demonstrated in studies where RIBEYE was efficiently co-immunoprecipitated with ArfGAP3 using specific antisera but not pre-immune serum .

How does ARFGAP3 localize relative to synaptic ribbons in photoreceptor synapses?

ARFGAP3 demonstrates specific localization patterns at photoreceptor ribbon synapses. Immunofluorescence studies reveal that ARFGAP3 is strongly enriched in the outer plexiform layer (OPL) of the retina where photoreceptor ribbon synapses are located . Super-resolution structured illumination microscopy (SR-SIM) has shown that ARFGAP3 immunosignals significantly overlap with RIBEYE, the major component of synaptic ribbons, indicating that ARFGAP3 is an integral component of the synaptic ribbon complex . Interestingly, detailed SR-SIM analyses revealed that the ARFGAP3 immunosignal is slightly shifted toward the inner nuclear layer compared to the RIBEYE immunosignal, suggesting ARFGAP3 may be preferentially localized toward the base of the synaptic ribbon . ARFGAP3 also shows strong colocalization with bassoon, an active zone protein located at the arciform density that anchors the base of the synaptic ribbon . When designing localization experiments, researchers should consider these subtle spatial relationships and employ high-resolution imaging techniques for accurate interpretation of ARFGAP3 distribution patterns.

How can antibody specificity be validated for ARFGAP3 localization studies?

Validating antibody specificity is crucial for reliable ARFGAP3 localization studies. Multiple independent validation approaches should be employed, including:

• Use of multiple antibodies against different epitopes of ARFGAP3: Studies have utilized two different polyclonal antibodies directed against distinct portions of the C-terminus of ARFGAP3 (ArfGAP Cterm2 and ArfGAP Cterm3), which yielded identical immunolabeling patterns, confirming specificity .

• Pre-absorption controls: Specificity can be confirmed by pre-absorbing the antibody with the ARFGAP3-GST fusion protein used as the immunogen. In validated studies, pre-absorption with the specific fusion protein blocked immunosignals, while pre-absorption with GST alone did not affect antibody binding .

• Western blot validation: Antibodies should detect a single major band at the expected molecular weight (~55 kDa in crude bovine retinal homogenates) that is absent in pre-immune serum controls and can be specifically blocked by the ArfGAP3-GST fusion protein .

• Affinity purification: For critical applications, affinity-purified antibodies can provide enhanced specificity. Techniques using fusion protein-loaded nitrocellulose strips have been successfully employed for ArfGAP3 antibody purification .

What is known about the interaction between ARFGAP3 and RIBEYE?

ARFGAP3 interacts with RIBEYE, a major component of synaptic ribbons, through multiple validated protein-protein interaction mechanisms. Fusion protein pull-down assays have demonstrated that the ArfGAP domain (AGD) of ArfGAP3 binds to the RIBEYE(B) domain . This interaction has been confirmed using both MBP-tagged and GST-tagged fusion proteins with switched tags, consistently showing strong binding affinity with typically >30% of input RIBEYE(B) binding to immobilized ArfGAP3 . The interaction is NAD(H)-dependent and redox-sensitive, with NADH being more efficient than oxidized NAD+ in promoting ArfGAP3-RIBEYE binding . Importantly, RIBEYE competes with the GTP-binding protein Arf1 for binding to ArfGAP3, suggesting a potential regulatory mechanism whereby RIBEYE binding to ArfGAP3 could prevent inactivation of Arf1 . When designing experiments to study this interaction, researchers should consider the redox state of the experimental system and include appropriate NADH/NAD+ controls to accurately assess binding dynamics.

How does ARFGAP3 function in endocytic vesicle trafficking at ribbon synapses?

ARFGAP3, though well-characterized as a regulator of vesicle formation at the Golgi apparatus, also plays a critical role in endocytic vesicle trafficking at ribbon synapses. Functional studies have demonstrated that overexpression of ArfGAP3 in photoreceptors strongly inhibits endocytotic uptake of FM1-43, indicating its importance in endocytic processes . The interaction between ArfGAP3 and RIBEYE appears to be functionally relevant for endocytic trafficking, potentially through regulation of Arf1 activity . RIBEYE competes with Arf1 for binding to ArfGAP3, suggesting that the synaptic ribbon can control Arf1 function through this competitive binding mechanism . When investigating endocytic trafficking at ribbon synapses, researchers should consider that ArfGAP3 is enriched at the base of the synaptic ribbon in close proximity to both RIBEYE and active zone proteins like bassoon . This strategic localization suggests it may function at a critical interface between exocytic and endocytic zones of these tonically active synapses with intense vesicle traffic .

What controls should be implemented when studying ARFGAP3 using immunological methods?

When studying ARFGAP3 using immunological methods, implementing appropriate controls is essential for generating reliable and interpretable data:

• Pre-immune serum controls: In immunoprecipitation and immunohistochemistry experiments, pre-immune serum should be used as a negative control to determine baseline non-specific binding. Studies have shown that pre-immune serum fails to immunoprecipitate RIBEYE and ArfGAP3, confirming the specificity of the interaction detected with immune serum .

• Antigen pre-absorption controls: Pre-absorption of antibodies with the specific ArfGAP3-GST fusion protein should abolish specific immunosignals, while pre-absorption with GST alone should not affect antibody binding. This control has successfully validated specificity in immunofluorescence studies of ArfGAP3 in retinal tissues .

• Multiple antibody validation: Using multiple antibodies directed against different epitopes of ArfGAP3 (such as ArfGAP3 Cterm2 and ArfGAP3 Cterm3) that produce identical immunolabeling patterns provides strong evidence for specificity .

• Cell/tissue positive controls: Include validated positive control samples such as HepG2 cells for Western blot and immunoprecipitation, mouse brain tissue for Western blot, human breast cancer tissue for immunohistochemistry, and A431 or HepG2 cells for immunofluorescence applications .

What are the challenges in electron microscopic localization of ARFGAP3?

Despite successful localization of ARFGAP3 using immunofluorescence and super-resolution microscopy, electron microscopic localization presents significant technical challenges. Multiple antibodies against ARFGAP3, including both laboratory-generated and commercially available ones, have been unsuccessful in electron microscopic applications using both pre-embedding and post-embedding procedures with immunogold and immunoperoxidase-based techniques . Specifically, the antibodies ArfGAP3Cterm2 and ArfGAP3Cterm3, while effective for Western blotting and immunofluorescence microscopy, did not work at the electron microscopic level with available laboratory procedures . Commercial antibodies from multiple sources (Antibody Verify, Novus Biologicals, Sigma, and Biomol) similarly failed in postembedding immunogold electron microscopy . This limitation suggests that the epitopes recognized by these antibodies may be particularly sensitive to the stronger fixation and processing required for electron microscopy. Researchers attempting ultrastructural localization of ARFGAP3 should consider alternative approaches such as epitope tagging combined with correlative light and electron microscopy, or development of antibodies specifically designed for preservation of epitopes during EM processing.