EXOC7 Antibody, FITC conjugated

What is EXOC7 and what cellular functions is it involved in?

EXOC7 (Exocyst Complex Component 7), also known as Exo70, is a subunit of the exocyst complex that plays a critical role in vesicle trafficking and membrane fusion. The exocyst complex is evolutionarily conserved from yeast to mammals and comprises eight subunits: Sec3, Sec5, Sec6, Sec8, Sec10, Sec15, Exo70, and Exo84 . EXOC7 specifically functions in the docking of exocytic vesicles with fusion sites on the plasma membrane. In adipocytes, it plays a crucial role in targeting SLC2A4 (GLUT4) vesicles to the plasma membrane in response to insulin stimulation, potentially directing these vesicles to precise fusion sites . Additionally, EXOC7 is required for neuron survival and plays an essential role in cortical development . Recent research using inducible CRISPR knockout systems has demonstrated that EXOC7 is critical for insulin-stimulated GLUT4 exocytosis in adipocytes .

What applications are most suitable for EXOC7 antibodies with fluorescent conjugation?

EXOC7 antibodies with fluorescent conjugation like FITC are particularly valuable for applications requiring direct visualization of the protein. Western blotting (WB) can be performed with EXOC7 antibodies at dilutions of approximately 1:1000, as demonstrated with multiple commercially available antibodies . For immunocytochemistry and immunofluorescence (ICC/IF) applications, EXOC7 antibodies typically perform well at dilutions ranging from 1:100 to 1:500, allowing visualization of subcellular localization patterns . Immunoprecipitation (IP) is another suitable application at approximately 1:100 dilution . When using FITC-conjugated antibodies specifically, flow cytometry (FC) applications are also feasible, typically at dilutions between 1:50 and 1:200 . Fluorescently-tagged EXOC7 antibodies are particularly valuable for co-localization studies with other vesicular trafficking components.

How should researchers validate the specificity of an EXOC7 antibody?

Validating EXOC7 antibody specificity requires a multi-faceted approach. First, perform Western blot analysis using target tissue or cell lysates (like rat brain or HeLa cells) to confirm detection of the expected molecular weight band (approximately 75-83 kDa) . Include positive controls such as HeLa whole cell lysate, which has been documented to show consistent EXOC7 expression . For definitive validation, implement genetic approaches such as the inducible CRISPR knockout system described for EXOC7, which can demonstrate antibody specificity through loss of signal after gene deletion . When validating for immunofluorescence applications, compare staining patterns with published localization data and perform blocking peptide experiments to confirm signal specificity. Additionally, use multiple antibodies targeting different epitopes of EXOC7 to corroborate localization patterns and ensure consistent results across different detection methods.

What are the optimal storage conditions for maintaining FITC-conjugated antibody performance?

FITC-conjugated antibodies require specific storage conditions to maintain fluorophore integrity and antibody performance. Store FITC-conjugated antibodies at -20°C in the dark to prevent photobleaching of the fluorophore . The storage buffer typically contains a cryoprotectant such as glycerol (often at 50%) to prevent freeze-thaw damage, physiological buffer like PBS to maintain pH stability, and stabilizers such as BSA (approximately 0.75%) to prevent protein degradation . It's critical to avoid repeated freeze-thaw cycles that can degrade both the antibody and the conjugated fluorophore. Never aliquot certain antibody formulations as specified by manufacturers . For working solutions, prepare only the necessary amount needed for immediate use and store at 4°C protected from light for up to one week. For long-term storage beyond the manufacturer's recommended shelf life, validate antibody performance before use in critical experiments.

What controls should be included when using FITC-conjugated EXOC7 antibodies?

When using FITC-conjugated EXOC7 antibodies, comprehensive controls are essential to ensure experimental validity. Include a negative control using an isotype-matched FITC-conjugated antibody of irrelevant specificity to assess background fluorescence and non-specific binding. Positive controls should include samples with known EXOC7 expression patterns, such as HeLa cells or rat brain tissue, which have been well-characterized . For knockout validation, compare staining between wild-type cells and those with EXOC7 gene deletion using inducible CRISPR systems . When performing multicolor immunofluorescence, include single-color controls to assess spectral overlap and facilitate compensation. Autofluorescence controls (unstained samples) help distinguish between specific signal and intrinsic sample fluorescence. Additionally, include absorption controls where the primary antibody is pre-incubated with recombinant EXOC7 protein before staining, which should result in signal reduction if the antibody is specific.

How can researchers optimize FITC-conjugated antibody staining for investigating EXOC7 dynamics during vesicle trafficking?

Optimizing FITC-conjugated antibody staining for EXOC7 dynamics requires several technical considerations. Begin with fixation method selection—for EXOC7, methanol fixation has shown good results in neuroendocrine PC12 cells, with overnight incubation at 4°C yielding optimal visualization . For paraformaldehyde fixation, such as that used with A431 cells, a 1:200 dilution has been effective . When tracking dynamic processes like insulin-stimulated GLUT4 trafficking, implement pulse-chase experiments with timed fixation points following stimulation. Enhance signal detection by implementing signal amplification techniques such as tyramide signal amplification if direct FITC conjugation provides insufficient signal strength. For co-localization studies with vesicular markers, ensure proper controls for bleed-through and employ high-resolution imaging techniques such as structured illumination microscopy or stimulated emission depletion microscopy to resolve closely associated structures beyond the diffraction limit. Quantify co-localization using established metrics such as Pearson's correlation coefficient, and analyze trafficking dynamics using time-lapse imaging combined with photoactivatable or photoconvertible EXOC7 fusion proteins.

What approaches can address potential photobleaching issues when using FITC-conjugated antibodies in live-cell EXOC7 imaging?

Addressing photobleaching when using FITC-conjugated antibodies for EXOC7 imaging requires multiple technical strategies. First, optimize imaging parameters by reducing excitation intensity, minimizing exposure times, and increasing detector sensitivity (using EMCCDs or sCMOS cameras with high quantum efficiency). Implement anti-fade reagents specifically formulated for live-cell imaging that are compatible with cell viability. Consider the use of oxygen scavenger systems such as glucose oxidase/catalase or OxyFluor to reduce reactive oxygen species that contribute to fluorophore degradation. For extended imaging sessions, employ computational approaches such as bleach correction algorithms during post-processing. If FITC photobleaching remains problematic despite these measures, consider alternative fluorophores with greater photostability such as Alexa Fluor 488, which offers similar spectral properties (excitation maximum near 491nm, emission maximum near 516nm) but superior resistance to photobleaching compared to FITC . For particularly challenging live-cell applications, implement advanced imaging modalities such as spinning disk confocal microscopy that minimize photobleaching through reduced excitation duration per pixel.

How should researchers analyze EXOC7 localization changes during insulin stimulation using quantitative immunofluorescence?

Analyzing EXOC7 localization changes during insulin stimulation requires rigorous quantitative approaches. Establish a time-course experiment with multiple fixation points following insulin stimulation (typically 0, 5, 10, 15, 30 minutes) in appropriate cell models such as adipocytes, where EXOC7 plays a crucial role in insulin-stimulated GLUT4 trafficking . Utilize high-resolution confocal microscopy with z-stacking to capture the full cellular volume. Implement automated image analysis pipelines to quantify EXOC7 redistribution, measuring parameters such as distance from plasma membrane, co-localization with membrane markers, and formation of punctate structures. For precise quantification, employ line-scan analysis across the cell periphery to generate fluorescence intensity profiles before and after insulin stimulation. Calculate the plasma membrane-to-cytosol ratio of EXOC7 signal intensity as a metric of translocation. To validate functional significance, perform parallel experiments with EXOC7 knockout models created using inducible CRISPR systems, which have demonstrated that EXOC7 is critical for insulin-stimulated GLUT4 exocytosis . Incorporate co-labeling with phospho-specific antibodies against insulin signaling components to correlate EXOC7 redistribution with activation of specific signaling pathways.

What methodological approaches can differentiate between specific EXOC7 staining and autofluorescence when using FITC-conjugated antibodies?

Differentiating specific EXOC7 staining from autofluorescence requires multiple methodological controls and techniques. First, always include unstained samples to establish baseline autofluorescence levels in your specific cell type or tissue. Implement spectral unmixing algorithms during image acquisition or post-processing to separate overlapping fluorescence signatures based on their distinct spectral profiles. Autofluorescence typically exhibits broader emission spectra than FITC, which has a relatively narrow emission peak at 516nm . Consider time-resolved fluorescence microscopy techniques that exploit the difference in fluorescence lifetime between specific antibody-conjugated FITC (typically 3-4 ns) and endogenous autofluorescent molecules (often <2 ns). For tissues with high autofluorescence, pretreat sections with Sudan Black B or commercial autofluorescence quenching reagents. Compare staining patterns between multiple EXOC7 antibodies recognizing different epitopes to confirm signal specificity—consistent localization patterns across different antibodies strongly suggest specific rather than artifactual staining. Additionally, include genetic controls such as EXOC7 knockdown or knockout samples, which should show reduction or elimination of specific staining while autofluorescence remains unchanged .

How can researchers troubleshoot weak signal intensity when using FITC-conjugated antibodies for detecting endogenous EXOC7 levels?

Troubleshooting weak EXOC7 signal requires systematic optimization of multiple parameters. First, evaluate fixation methods—methanol fixation has proven effective for EXOC7 detection in PC12 cells with overnight primary antibody incubation at 4°C , while paraformaldehyde fixation works well for A431 cells . Optimize antibody concentration through titration experiments—standard dilutions range from 1:100 to 1:500 for immunofluorescence, but higher concentrations may be necessary for detecting low-abundance endogenous protein . Implement epitope retrieval methods such as heat-induced epitope retrieval or enzymatic digestion if epitope masking is suspected. Consider signal amplification approaches such as tyramide signal amplification, which can increase sensitivity by 10-100 fold compared to direct detection. For applications requiring highest sensitivity, switch from direct FITC-conjugated primary antibodies to a two-step approach using unconjugated primary antibody followed by highly cross-adsorbed FITC-conjugated secondary antibody, which provides signal amplification as multiple secondary antibodies bind each primary antibody . Optimize image acquisition parameters by increasing exposure time (while monitoring photobleaching), adjusting detector gain, and using objectives with higher numerical aperture to collect more emitted light.

What experimental design is recommended for studying EXOC7's role in insulin-stimulated GLUT4 trafficking using antibody-based approaches?

For studying EXOC7's role in insulin-stimulated GLUT4 trafficking, implement a comprehensive experimental design with appropriate controls. Establish a cell model system using adipocytes or muscle cells that robustly express both EXOC7 and GLUT4. Create an inducible EXOC7 knockout system using CRISPR technology as described in recent literature, allowing for temporal control of EXOC7 depletion to avoid confounding effects on differentiation . Design a time-course experiment with insulin stimulation (typically 100nM) for various durations (0, 5, 10, 15, 30 minutes). Employ dual immunofluorescence labeling with antibodies against EXOC7 (using appropriate dilutions of 1:100-1:200) and GLUT4 to track their dynamic co-localization. Include quantitative plasma membrane fractionation experiments to biochemically validate microscopy findings. For functional validation, perform glucose uptake assays in control versus EXOC7-depleted cells. Incorporate total internal reflection fluorescence (TIRF) microscopy to specifically visualize events at the plasma membrane with high signal-to-noise ratio. Implement live-cell imaging using photoactivatable or photoconvertible fusion proteins to track the dynamic behavior of EXOC7-containing vesicles in real-time. Finally, conduct rescue experiments by reintroducing wild-type or mutant EXOC7 variants to determine critical domains required for function.

How should researchers optimize co-immunoprecipitation protocols when investigating EXOC7 interactions with other exocyst complex components?

Optimizing co-immunoprecipitation (co-IP) protocols for EXOC7 interactions requires careful consideration of experimental conditions. Select an appropriate EXOC7 antibody with validated IP capability, such as the EXOC7 (E4W6R) Rabbit mAb which has been successfully used at 1:100 dilution for immunoprecipitation . Choose lysis buffers that preserve protein-protein interactions while effectively solubilizing membrane-associated complexes—typically containing 1% NP-40 or 0.5% Triton X-100, 150mM NaCl, 50mM Tris-HCl (pH 7.4), and protease inhibitor cocktail. Include phosphatase inhibitors if phosphorylation-dependent interactions are suspected. Optimize antibody-to-lysate ratios through titration experiments, typically starting with 1-5μg antibody per 500μg total protein. Pre-clear lysates with protein G beads to reduce non-specific binding. Carefully select wash conditions that remove non-specific interactions while preserving specific ones, typically using 3-5 washes with decreasing detergent concentrations. Validate results using reciprocal co-IPs with antibodies against different exocyst components. Include negative controls using isotype-matched irrelevant antibodies and positive controls using known interacting proteins. For challenging interactions, consider crosslinking approaches or proximity labeling techniques such as BioID or APEX as complementary methods to traditional co-IP. When analyzing results, the predicted molecular weight for EXOC7 is approximately 75-83 kDa .

How can researchers validate EXOC7 knockout models when studying the functional importance of this protein?

Validating EXOC7 knockout models requires a multi-faceted approach to confirm complete protein depletion and assess functional consequences. Implement Western blot analysis using validated EXOC7 antibodies at 1:1000 dilution to confirm protein depletion, comparing knockout samples with wild-type controls . For inducible knockout systems, perform time-course experiments to determine the kinetics of protein depletion following induction . Conduct immunofluorescence microscopy using FITC-conjugated or standard primary antibodies (at 1:100-1:200 dilution) with appropriate secondary detection to visually confirm loss of EXOC7 signal in knockout cells . Verify knockout at the genomic level using PCR and sequencing to confirm the intended genetic modification. For functional validation, assess established EXOC7-dependent processes such as insulin-stimulated GLUT4 trafficking in adipocytes using glucose uptake assays . Examine the formation and composition of remaining exocyst complexes through co-immunoprecipitation experiments with antibodies against other exocyst components. Perform rescue experiments by reintroducing wild-type EXOC7 to confirm that observed phenotypes are specifically due to EXOC7 depletion rather than off-target effects. For comprehensive validation, combine genetic approaches with pharmacological inhibition of pathways known to regulate EXOC7 function.

What strategies can minimize background fluorescence when using FITC-conjugated antibodies in complex tissue samples?

Minimizing background fluorescence in complex tissue samples requires implementing multiple optimization strategies. Begin with thorough sample preparation, including proper fixation—paraformaldehyde or methanol fixation has been successfully used with EXOC7 antibodies . Implement effective blocking procedures using a combination of serum (5-10%) matched to the secondary antibody host species, BSA (1-3%), and detergents like Triton X-100 (0.1-0.3%) to reduce non-specific binding. Consider adding mouse IgG blocking reagents when working with mouse tissues and mouse-derived primary antibodies. Treat samples with Sudan Black B (0.1-0.3% in 70% ethanol) or commercial autofluorescence quenchers to reduce endogenous tissue autofluorescence. Optimize antibody concentrations through careful titration experiments—while standard dilutions for immunofluorescence range from 1:100-1:500, tissue work may require further optimization . Include extensive washing steps (minimum 3 x 5 minutes) with agitation using PBS containing 0.05-0.1% Tween-20. When designing multi-color experiments, carefully select fluorophore combinations to minimize spectral overlap with tissue autofluorescence, which often occurs in the FITC/green channel. Consider using confocal microscopy with spectral detection to separate specific signal from autofluorescence based on their distinct spectral signatures.

How can super-resolution microscopy techniques enhance EXOC7 localization studies beyond conventional immunofluorescence?

Super-resolution microscopy offers significant advantages for EXOC7 localization studies by overcoming the diffraction limit of conventional microscopy. Structured Illumination Microscopy (SIM) provides approximately 100nm resolution, enabling more precise visualization of EXOC7 distribution at the plasma membrane and its association with vesicular structures. For optimal SIM imaging, use highly specific EXOC7 antibodies at slightly higher concentrations than conventional immunofluorescence (approximately 1:50-1:100 dilution) to ensure sufficient signal intensity . Stimulated Emission Depletion (STED) microscopy offers even greater resolution (~30-50nm) and is particularly valuable for resolving individual EXOC7-containing structures at vesicle docking sites. Single-molecule localization techniques such as STORM or PALM provide the highest resolution (~10-20nm) and are ideal for precise mapping of EXOC7 within multiprotein complexes, though these approaches require special fluorophores and sample preparation. For any super-resolution application, sample preparation is critical—use thin sections (≤10μm), optimal fixation protocols, and mounting media specifically formulated for super-resolution techniques. Implement appropriate image processing algorithms for each method, and include resolution standards to validate the achieved resolution. These techniques can reveal previously unobservable details of EXOC7 organization during vesicle trafficking and membrane fusion events.

What approaches combine EXOC7 protein detection with RNA analysis for comprehensive expression studies?

Combining EXOC7 protein detection with RNA analysis provides a comprehensive understanding of expression regulation. Implement fluorescence in situ hybridization (FISH) for EXOC7 mRNA combined with immunofluorescence using FITC-conjugated EXOC7 antibodies to simultaneously visualize mRNA and protein distributions within the same samples. For this combined protocol, perform the RNA hybridization steps first, followed by immunodetection using EXOC7 antibodies at appropriate dilutions (typically 1:100-1:200) . Single-cell approaches such as CITE-seq can simultaneously measure EXOC7 protein (using antibody-based detection) and mRNA (through single-cell RNA-seq) in thousands of individual cells, revealing cell-to-cell variation and potential post-transcriptional regulation. For temporal dynamics, use nascent RNA labeling techniques such as EU incorporation combined with EXOC7 immunofluorescence to assess the time course of RNA synthesis to protein expression. When studying EXOC7 regulation in knockout models, combine RT-qPCR for mRNA quantification with Western blotting (using antibodies at 1:1000 dilution) for protein analysis to distinguish between transcriptional and post-transcriptional effects . For tissues, implement slide-seq or Visium spatial transcriptomics alongside immunohistochemistry on adjacent sections to correlate spatial patterns of mRNA and protein expression across complex tissue architecture.