VAMP4 Antibody

What is VAMP4 and what cellular functions does it perform?

VAMP4 (vesicle-associated membrane protein 4) is a member of the VAMP/synaptobrevin family and functions as a SNARE protein involved in membrane trafficking. It plays essential roles in various cellular processes including trans-Golgi network-to-endosome transport, maintenance of Golgi structure, and regulation of secretory pathways . Recent studies have demonstrated that VAMP4 participates in multiple SNARE complexes, including those with STX6 for TGN-to-endosome anterograde transport and with STX16, VTI1A, and STX6 for endosome-to-TGN retrograde trafficking . Additionally, VAMP4 forms complexes with STX7, STX8, and VTI1B on lysosomes to facilitate the fusion of insulin secretory granules with lysosomes, thereby regulating insulin levels .

What applications are VAMP4 antibodies suitable for?

VAMP4 antibodies have been validated for multiple experimental applications through rigorous testing. The primary applications include:

These applications have been validated across multiple tissue types and cell lines, with particularly strong signals observed in brain tissue, testis tissue, and neuronal cell lines . It is strongly recommended to optimize antibody concentrations for each experimental system to achieve optimal signal-to-noise ratios.

What species reactivity can researchers expect from VAMP4 antibodies?

Based on extensive validation data, commercially available VAMP4 antibodies show confirmed reactivity with human, mouse, and rat samples . The high sequence conservation of VAMP4 across these species allows for cross-reactivity, making these antibodies versatile tools for comparative studies. When selecting an antibody for your research, verify the specific epitope recognition, as some antibodies target conserved regions while others may be species-specific. The polyclonal antibody (10738-1-AP) has been extensively validated in various applications across these species, particularly in neuronal and pancreatic tissues .

How can VAMP4 antibodies be used to investigate insulin secretion pathways?

VAMP4 plays a critical regulatory role in insulin secretion, making VAMP4 antibodies valuable tools for investigating diabetes and related metabolic disorders. Recent knockout studies have revealed that VAMP4 deficiency causes significant physiological changes in pancreatic β cells, including increased blood insulin levels, accumulated intracellular (pro)insulin, and elevated insulin release . To effectively study these pathways:

• Implement co-localization studies using VAMP4 antibodies (IF/ICC at 1:200-1:800 dilution) alongside insulin and proinsulin markers to assess intracellular distribution in β cells.

• Utilize immunoprecipitation (0.5-4.0 μg antibody) to capture VAMP4-interacting proteins, particularly other SNARE complex components like STX7, STX8, and VTI1B, which mediate the fusion of immature insulin secretory granules with lysosomes.

• Compare wild-type and VAMP4-deficient cells using quantitative immunofluorescence to measure relative insulin and proinsulin levels. In VAMP4 knockout cells, proinsulin levels were 4.5-fold higher and insulin levels 1.7-fold higher than in wild-type cells .

• Conduct glucose-stimulated insulin secretion assays in conjunction with VAMP4 immunostaining to correlate VAMP4 localization with functional insulin release dynamics.

These approaches can reveal how VAMP4 controls insulin homeostasis through its involvement in targeting secretory granules to lysosomes, providing critical insights into β cell physiology and potential therapeutic targets.

What methodological considerations are important when using VAMP4 antibodies to study Golgi structure?

VAMP4 is essential for maintaining Golgi ribbon structure, as demonstrated by RNAi-mediated depletion studies. When investigating this function, several methodological considerations are crucial:

• Implement time-course experiments when conducting VAMP4 knockdown studies, as protein depletion may not be evident until 48-72 hours post-transfection. Western blotting analysis should be used to confirm depletion efficiency before assessing Golgi morphology .

• Employ dual immunofluorescence using VAMP4 antibodies (1:200-1:800) alongside Golgi markers (e.g., GM130, TGN46) to evaluate Golgi fragmentation and organization. VAMP4 depletion has been shown to result in Golgi fragmentation while maintaining the juxtanuclear position of fragments .

• Include appropriate controls in knockdown experiments to distinguish between specific VAMP4-related effects and non-specific consequences of experimental manipulation.

• Consider using super-resolution microscopy techniques (STED, STORM) with VAMP4 antibodies to capture subtle changes in Golgi morphology that might be missed by conventional confocal microscopy.

• Implement rescue experiments by expressing siRNA-resistant VAMP4 constructs followed by immunostaining to confirm that observed Golgi fragmentation is directly attributable to VAMP4 depletion rather than off-target effects.

The effects of VAMP4 on Golgi structure are distinct from other VAMP proteins, highlighting the importance of isoform-specific antibodies for accurate localization and functional studies.

How can researchers optimize co-immunoprecipitation protocols to study VAMP4-SNARE complexes?

VAMP4 forms distinct SNARE complexes in different cellular compartments, making co-immunoprecipitation (co-IP) a valuable approach for studying these interactions. Based on published methodologies, researchers should consider:

• Selection of detergent conditions is critical for preserving SNARE complexes. Use mild non-ionic detergents (0.5-1% Triton X-100 or 1% CHAPS) rather than harsh ionic detergents that may disrupt protein-protein interactions.

• Implement specific buffer compositions based on the target complex. For example, when investigating VAMP4-Syntaxin4 interactions, researchers should note that this complex was not detected in co-IP experiments, while VAMP4-Syntaxin16 complexes were readily observable .

• Consider using cross-linking reagents (DSP, formaldehyde) before lysis to stabilize transient interactions, particularly for dynamic SNARE complexes.

• Optimize antibody concentrations for immunoprecipitation. For VAMP4, using 0.5-4.0 μg antibody per 1.0-3.0 mg of total protein lysate has proven effective .

• Include appropriate controls: IgG control, reciprocal IPs (pulling down with anti-Syntaxin and blotting for VAMP4, and vice versa), and analysis of different stimulation conditions (e.g., insulin stimulation when studying GLUT4 trafficking) .

These methodological refinements will enhance detection sensitivity and specificity when investigating VAMP4's participation in various SNARE complexes, including those with STX7, STX8, and VTI1B for lysosomal fusion or with STX16 for endosome-to-TGN trafficking.

What approaches can resolve contradictory data regarding VAMP4 localization and function?

Reconciling contradictory findings regarding VAMP4 localization and function requires rigorous methodological approaches:

• Implement quantitative colocalization analysis rather than simple visual assessment. Studies by Zhao et al. showed 20-50% overlap between different VAMPs and GLUT4 in membrane fractions, but this approach may not account for different expression levels of various VAMPs in the same vesicle .

• Use super-resolution microscopy coupled with precise colocalization algorithms to distinguish between true colocalization and proximity due to limited optical resolution.

• Employ complementary techniques beyond immunofluorescence, such as subcellular fractionation followed by Western blotting, to validate localization patterns.

• Consider the possibility of cell-type specific differences in VAMP4 function. While VAMP4 forms complexes with Syntaxin16 in adipocytes, it associates with different syntaxins in other cell types (e.g., STX5 and SNAP23 for lipid droplet fusion) .

• Design experiments that account for potential compensatory mechanisms. Studies with VAMP4 knockout models suggest that other trafficking routes may compensate for VAMP4 deficiency, which could explain seemingly contradictory results .

• Utilize proximity labeling approaches (BioID, APEX) with VAMP4 as bait to identify the protein's neighborhood in different cellular contexts, potentially revealing context-dependent functions.

By implementing these methodological refinements, researchers can better resolve the seemingly contradictory roles of VAMP4 across different cell types and experimental systems.

How can researchers quantitatively assess VAMP4 expression levels in different cell types?

Accurate quantification of VAMP4 expression is essential for comparative studies across cell types and experimental conditions. The following methodological approaches are recommended:

• Implement quantitative Western blotting using recombinant VAMP4 protein standards. As demonstrated in studies with 3T3-L1 adipocytes, comparing immunoblot signals from total membrane samples against known amounts of recombinant protein allows precise estimation of VAMP4 abundance .

• For cellular quantification, correlate protein measurements with cell counts (e.g., using propidium iodide staining of nuclei) to determine VAMP4 content per cell. Previous studies found that a 10-cm dish of 3T3-L1 adipocytes (approximately 10^7 cells) yielded about 2.5 mg of protein .

• Apply flow cytometry using permeabilized cells and fluorescently-labeled VAMP4 antibodies for high-throughput quantification across cell populations.

• Use quantitative PCR to measure VAMP4 mRNA levels, though post-transcriptional regulation may result in discrepancies between mRNA and protein abundance.

• Implement SILAC or other mass spectrometry-based quantification methods for absolute quantification of VAMP4 in complex protein mixtures.

When interpreting quantitative data, researchers should consider potential caveats such as differential antibody affinity for recombinant versus endogenous proteins, and the impact of post-translational modifications on antibody recognition.

What strategies can address non-specific binding when using VAMP4 antibodies?

Non-specific binding is a common challenge when working with VAMP4 antibodies, particularly in applications like immunofluorescence and immunohistochemistry. To minimize this issue:

• Optimize blocking conditions by testing different blocking agents (BSA, normal serum, commercial blocking buffers) at various concentrations (1-5%) and incubation times (30 minutes to overnight).

• Implement stringent washing steps using PBS-T (0.1-0.3% Tween-20) or TBS-T to remove weakly bound antibodies while preserving specific signals.

• Titrate primary antibody concentrations, starting with the recommended range (1:200-1:800 for IF/ICC, 1:250-1:1000 for IHC) and adjusting based on signal-to-noise ratio .

• Include appropriate controls: (a) no primary antibody control, (b) isotype control, (c) pre-absorption control using recombinant VAMP4 protein, and (d) VAMP4 knockout/knockdown samples when available.

• For tissues requiring antigen retrieval, compare TE buffer (pH 9.0) with citrate buffer (pH 6.0) to determine optimal conditions for specific VAMP4 detection .

• Consider using monoclonal antibodies if polyclonal antibodies show high background, though this may come at the cost of reduced sensitivity.

By systematically optimizing these parameters, researchers can significantly improve the specificity of VAMP4 detection while minimizing background signals.

How can researchers validate VAMP4 antibody specificity in knockout/knockdown systems?

Rigorous validation of VAMP4 antibody specificity using genetic models is essential for reliable research outcomes:

• Implement CRISPR/Cas9-mediated VAMP4 knockout models as gold-standard controls. Complete VAMP4 deletion should result in absence of signal across all applications (Western blot, IF, IHC) .

• For RNAi-based approaches, monitor the time-dependent reduction in VAMP4 expression, as studies have shown that significant depletion may take 48-72 hours post-transfection .

• Use quantitative Western blotting to assess knockdown efficiency, with successful RNAi approaches reducing VAMP4 protein levels to 6-32% of control levels .

• Implement rescue experiments by expressing siRNA-resistant VAMP4 constructs to confirm that phenotypes are directly attributable to VAMP4 depletion rather than off-target effects.

• In immunofluorescence experiments with mixed cultures of wild-type and knockout cells, VAMP4-specific antibodies should show clear differential staining patterns, providing an internal control for specificity .

• Consider using multiple antibodies targeting different epitopes of VAMP4 to confirm specificity, as agreement between different antibodies strengthens validation.

These approaches provide comprehensive validation of antibody specificity, ensuring that observed signals genuinely represent VAMP4 rather than cross-reactive proteins.

What methods best capture the dynamic trafficking of VAMP4 in living cells?

To investigate the dynamic behavior of VAMP4 in real-time:

• Implement live-cell imaging using VAMP4-fluorescent protein fusions (GFP, mCherry) in conjunction with markers for different cellular compartments.

• Consider using the RUSH system (Retention Using Selective Hooks) to synchronize VAMP4 trafficking through the secretory pathway, allowing precise temporal analysis of its movement.

• Apply FRAP (Fluorescence Recovery After Photobleaching) or photoactivation approaches to measure VAMP4 mobility and exchange rates between different cellular compartments.

• Implement super-resolution live-cell imaging techniques (lattice light sheet, TIRF-SIM) to capture VAMP4-positive vesicle dynamics with high spatial and temporal resolution.

• Complement imaging with biochemical approaches such as surface biotinylation or subcellular fractionation at different time points to track VAMP4 redistribution in response to stimuli.

These methodologies can reveal how VAMP4 dynamically regulates processes such as insulin secretory granule maturation and sorting to lysosomes, providing insights into its physiological functions that static approaches might miss.

How do post-translational modifications affect VAMP4 antibody detection?

VAMP4 undergoes various post-translational modifications that can impact antibody recognition:

• Consider the epitope location of your antibody relative to known modification sites. Antibodies targeting regions susceptible to phosphorylation, ubiquitination, or SUMOylation may show variable detection depending on the modification status.

• Implement phosphatase treatment of samples prior to Western blotting to determine if phosphorylation affects antibody recognition.

• Use inhibitors of specific modifications (phosphatase inhibitors, deubiquitinase inhibitors) to stabilize modified forms for detection.

• Consider using modification-specific antibodies in conjunction with general VAMP4 antibodies to correlate modification status with localization or function.

• For quantitative studies, be aware that the observed molecular weight of VAMP4 (18 kDa) differs from the calculated weight (16 kDa), likely due to post-translational modifications .

Understanding how modifications affect antibody detection is particularly important when studying VAMP4's dynamic roles in membrane trafficking, where modifications may regulate its incorporation into different SNARE complexes.