VPS16 Antibody, FITC conjugated

What is VPS16 and what cellular functions does it regulate?

VPS16 (Vacuolar protein sorting 16 homolog) is an 839 amino acid protein with a molecular weight of approximately 95 kDa that functions as a crucial component of the Class C VPS protein complex, which includes VPS11, VPS18, and VPS33. This protein is predominantly located on the cytoplasmic side of membranes and is ubiquitously expressed across various tissues. VPS16 plays a fundamental role in vesicle-mediated protein sorting, which is essential for the proper segregation of intracellular molecules into distinct organelles. Specifically, it facilitates membrane docking and fusion reactions of late endosomes and lysosomes, making it vital for maintaining cellular homeostasis through proper trafficking of endocytic and biosynthetic proteins to the lysosomal compartments .

Disruptions in VPS16 function due to genetic mutations can lead to impaired lysosomal trafficking, potentially contributing to diseases such as Hermansky-Pudlak syndrome. The protein's conservation across species (from yeast to humans) underscores its evolutionary importance in fundamental cellular processes .

What are the key applications for VPS16 antibodies in research?

VPS16 antibodies are valuable tools for investigating vesicular trafficking pathways and lysosomal function through multiple experimental approaches:

These applications have been documented in numerous published studies, with at least 15 publications utilizing VPS16 antibodies for Western blotting and 9 publications for immunofluorescence applications .

What types of samples have been successfully used with VPS16 antibodies?

VPS16 antibodies have demonstrated reactivity with samples from multiple species and tissue types:

Researchers should note that optimal antigen retrieval methods may vary by tissue type. For human liver tissue samples, antigen retrieval with TE buffer pH 9.0 is suggested, though citrate buffer pH 6.0 may serve as an alternative .

What is the optimal protocol for conjugating FITC to VPS16 antibodies?

The optimal protocol for conjugating FITC to VPS16 antibodies follows established methods for antibody-fluorophore conjugation with specific parameters to maximize efficiency:

• Purification of Antibody: Start with a highly purified IgG preparation, preferably obtained through DEAE Sephadex chromatography, to ensure consistent conjugation results .

• Reaction Conditions: Optimal FITC conjugation occurs under these parameters:

  • Reaction temperature: Room temperature (20-25°C)

  • pH: 9.5 (carbonate-bicarbonate buffer)

  • Initial protein concentration: 25 mg/ml

  • Reaction time: 30-60 minutes

  • FITC quality: High-purity grade fluorescein isothiocyanate

• Purification of Conjugate: After the reaction, separate optimally labeled antibodies from under- and over-labeled proteins using gradient DEAE Sephadex chromatography. This step is crucial for obtaining conjugates with optimal fluorescein/protein (F/P) ratios .

Studies have demonstrated that electrophoretically distinct IgG molecules generally have similar affinity for FITC, suggesting that the conjugation protocol can be applied uniformly across different antibody preparations .

How do the different epitope regions of VPS16 affect antibody performance?

Different commercial VPS16 antibodies target distinct epitope regions, which can significantly impact their performance in specific applications:

The choice of epitope region can significantly impact:

• Accessibility in different experimental conditions

• Potential for cross-reactivity with related proteins

• Recognition of post-translationally modified VPS16

• Performance in detecting different VPS16 isoforms

For FITC-conjugated applications, researchers should consider whether the labeling might interfere with epitope recognition, particularly for antibodies targeting regions that contain lysine residues, which are primary targets for FITC conjugation .

How can researchers assess and optimize the F/P ratio in FITC-conjugated VPS16 antibodies?

The fluorescein/protein (F/P) ratio is a critical parameter that determines the performance of FITC-conjugated antibodies. For optimal results with FITC-conjugated VPS16 antibodies:

• Measurement Methods:

  • Spectrophotometric determination: Measure absorbance at 280 nm (protein) and 495 nm (fluorescein)

  • Calculate the F/P ratio using established formulas that account for the contribution of fluorescein to the 280 nm absorption

• Optimal F/P Ratio:

  • For most immunofluorescence applications, an F/P ratio of 2-3 provides optimal results

  • Higher ratios may increase background fluorescence

  • Lower ratios may provide insufficient signal intensity

• Controlling F/P Ratio:

  • Adjust the initial FITC:protein molar ratio in the reaction mixture

  • Modify reaction time (30-60 minutes typically optimal)

  • Control pH (higher pH increases conjugation efficiency)

  • Fractionation techniques can separate conjugates with different F/P ratios

Studies have shown a correlation between the activity of antibodies in fluorescent techniques and precipitation methods, suggesting that properly conjugated FITC-VPS16 antibodies should retain their specificity and binding characteristics .

What fixation and permeabilization methods are optimal for immunofluorescence using FITC-conjugated VPS16 antibodies?

The choice of fixation and permeabilization methods significantly impacts the detection of VPS16 with FITC-conjugated antibodies in immunofluorescence applications:

For VPS16 detection, which is predominantly located on the cytoplasmic side of membranes, a recommended protocol includes:

• Fix cells with 4% paraformaldehyde for 15-20 minutes at room temperature

• Permeabilize with 0.1-0.2% Triton X-100 for 5-10 minutes

• Block with appropriate blocking buffer (e.g., 3-5% BSA or normal serum)

• Incubate with FITC-conjugated VPS16 antibody at the recommended dilution

• Wash thoroughly to remove unbound antibody

• Mount with anti-fade mounting medium to preserve FITC fluorescence

This approach balances adequate fixation with epitope preservation and has been successful in published immunofluorescence studies using VPS16 antibodies .

What are the critical controls for validating FITC-conjugated VPS16 antibody specificity?

Rigorous validation of FITC-conjugated VPS16 antibodies requires several controls to ensure specificity and reliability of results:

• Negative Controls:

  • Isotype control: FITC-conjugated non-specific antibody of the same isotype (e.g., FITC-conjugated rabbit IgG for polyclonal VPS16 antibodies or FITC-conjugated mouse IgG2a for monoclonal clones like 2F10)

  • Secondary antibody only control (for indirect immunofluorescence)

  • Cells known to lack or express very low levels of VPS16

• Positive Controls:

  • Cells with confirmed high VPS16 expression (e.g., HeLa, HepG2, or Jurkat cells)

  • Tissue samples with known VPS16 expression (e.g., human or mouse liver tissue)

• Specificity Validation:

  • Peptide competition/blocking experiments

  • Knockdown/knockout validation (documented in at least 3 publications)

  • Cross-validation with multiple antibodies targeting different VPS16 epitopes

  • Western blot correlation with immunofluorescence patterns

• Technical Validation:

  • Concentration-dependent signal assessment

  • Co-localization with established lysosomal/endosomal markers

  • Signal-to-background ratio optimization

Implementing these controls helps distinguish specific signals from artifacts and ensures the reliability of research findings when using FITC-conjugated VPS16 antibodies.

How can researchers optimize storage conditions to maintain FITC-conjugated VPS16 antibody activity?

Proper storage is crucial for maintaining the activity of FITC-conjugated VPS16 antibodies. FITC is susceptible to photobleaching and can degrade under suboptimal conditions:

For working solutions of FITC-conjugated VPS16 antibodies:

• Prepare fresh dilutions when possible

• Store diluted antibody at 4°C for short periods (1-7 days)

• Protect from light using amber tubes or by wrapping containers in aluminum foil

• Add stabilizing proteins (BSA) to diluted antibody solutions at 0.1-1%

• Document date of thawing and dilution

Proper storage conditions significantly impact the longevity and performance of FITC-conjugated antibodies in research applications .

How can researchers address weak or non-specific signals when using FITC-conjugated VPS16 antibodies?

Troubleshooting weak or non-specific signals requires a systematic approach:

For optimal results, manufacturers recommend titrating FITC-conjugated VPS16 antibodies in each testing system to obtain optimal results, as optimal concentrations may be sample-dependent .

What strategies can optimize multicolor immunofluorescence including FITC-conjugated VPS16 antibodies?

When incorporating FITC-conjugated VPS16 antibodies into multicolor imaging experiments:

• Fluorophore Selection:

  • Choose fluorophores with minimal spectral overlap with FITC (Ex: 490nm, Em: 525nm)

  • Compatible options include:

    • DAPI/Hoechst for nuclei (blue)

    • TRITC/Cy3 for second target (red)

    • APC/Cy5 for third target (far-red)

• Sequential Staining Protocol:

  • For co-staining VPS16 with other lysosomal/endosomal markers:
    a. Fix and permeabilize cells (4% PFA followed by 0.1% Triton X-100)
    b. Block with 3-5% BSA or normal serum
    c. Apply FITC-conjugated VPS16 antibody
    d. Wash thoroughly (3-5 times with PBS)
    e. Apply additional primary antibodies
    f. Add appropriate fluorophore-conjugated secondary antibodies
    g. Counterstain nucleus with DAPI
    h. Mount with anti-fade mounting medium

• Acquisition Optimization:

  • Capture single-color controls for compensation

  • Minimize exposure times to reduce photobleaching

  • Use sequential scanning rather than simultaneous acquisition

  • Acquire images in order from longest to shortest wavelength

• Image Analysis Considerations:

  • Apply appropriate background subtraction

  • Use co-localization plugins/software (e.g., JACoP in ImageJ)

  • Quantify VPS16 distribution relative to other markers

  • Consider 3D reconstruction for volumetric analysis

These strategies maximize signal quality and minimize cross-talk when investigating VPS16 localization and interactions with other cellular components.

How does VPS16 dysfunction contribute to human disease pathology?

VPS16 dysfunction has been implicated in several pathological conditions:

• Lysosomal Storage Disorders:

  • Disruptions in VPS16 function can lead to impaired lysosomal trafficking

  • This dysfunction has been linked to Hermansky-Pudlak syndrome, a rare autosomal recessive disorder characterized by oculocutaneous albinism, bleeding problems, and pulmonary fibrosis

  • FITC-conjugated VPS16 antibodies enable visualization of protein mislocalization in affected cells

• Neurodegenerative Diseases:

  • VPS16's role in the Class C VPS complex is essential for proper endolysosomal function

  • Endolysosomal dysfunction is increasingly recognized as a contributing factor in neurodegenerative conditions

  • FITC-labeled antibodies allow researchers to track VPS16 dynamics in neuronal models

• Cancer Research Applications:

  • Altered vesicular trafficking pathways contribute to cancer progression and therapeutic resistance

  • VPS16 antibodies have been validated in liver cancer tissue , providing tools for investigating its role in oncogenesis

  • Fluorescent labeling enables high-resolution imaging of trafficking defects in tumor cells

FITC-conjugated VPS16 antibodies provide valuable tools for investigating these disease connections through advanced imaging techniques, potentially leading to new therapeutic targets focused on restoring normal vesicular trafficking.

What emerging technologies can enhance research using FITC-conjugated VPS16 antibodies?

Several cutting-edge technologies can significantly enhance research using FITC-conjugated VPS16 antibodies:

• Super-resolution Microscopy:

  • STED (Stimulated Emission Depletion) microscopy can achieve resolution below 50 nm with FITC-labeled samples

  • PALM/STORM techniques can provide single-molecule localization of VPS16

  • These approaches enable detailed visualization of VPS16 distribution at the subcellular level

• Live-cell Imaging Applications:

  • Cell-permeable FITC-conjugated antibody fragments (Fab fragments)

  • Antibody internalization techniques for tracking VPS16 dynamics

  • Correlative light-electron microscopy for combining fluorescence with ultrastructural analysis

• High-content Screening:

  • Automated imaging platforms for screening genetic or pharmacological modulators of VPS16 function

  • Quantitative analysis of VPS16 distribution across multiple conditions

  • Identification of compounds that affect vesicular trafficking pathways

• Multi-omics Integration:

  • Combining FITC-VPS16 antibody imaging with proteomics data

  • Correlating VPS16 localization with transcriptomic profiles

  • Integrating imaging data with interactome analyses

These technologies extend the capabilities of traditional fluorescence microscopy, enabling more detailed and quantitative analysis of VPS16 function in both normal physiology and disease states.