VPS4B Antibody, FITC conjugated

What is VPS4B and what cellular functions does it regulate?

VPS4B (Vacuolar protein sorting-associated protein 4B) is an AAA-ATPase involved in the late steps of the endosomal multivesicular bodies (MVB) pathway. It recognizes membrane-associated ESCRT-III assemblies and catalyzes their disassembly, possibly in combination with membrane fission. This protein redistributes ESCRT-III components to the cytoplasm for further rounds of MVB sorting, enabling the degradation of membrane proteins like growth factor receptors, lysosomal enzymes, and lipids . VPS4B also functions in topologically equivalent membrane fission events including cytokinetic abscission and enveloped virus budding (particularly HIV-1 and other lentiviruses). Additionally, VPS4A/B are required for the exosomal release of proteins such as SDCBP, CD63, and syndecan .

What are the key specifications of commercial VPS4B Antibody, FITC conjugated?

VPS4B Antibody, FITC conjugated is typically a polyclonal antibody derived from rabbit hosts. The antibody specifications generally include:

• Applications: Western Blot (WB), Immunofluorescence on paraffin-embedded tissues (IF/IHC-P), Immunofluorescence on frozen tissues (IF/IHC-F), Immunocytochemistry (ICC), and ELISA

• Reactivity: Human, Mouse, Fish (with predicted reactivity to Rat, Cow, and Pig)

• Concentration: Usually supplied at 1μg/μl

• Source: KLH-conjugated synthetic peptide derived from human VPS4B (immunogen range: 301-400/444) or recombinant Human VPS4B protein (aa 1-117)

• Storage: Recommended at -20°C in an aqueous buffered solution containing components such as TBS (pH 7.4), BSA, Proclin300, and 50% Glycerol

How does FITC conjugation benefit experimental applications compared to unconjugated antibodies?

FITC (Fluorescein isothiocyanate) conjugation provides several experimental advantages:

• Direct detection without secondary antibodies, reducing experimental time and potential cross-reactivity issues

• Excitation/emission peaks at approximately 495nm/519nm, compatible with standard fluorescence microscopy filter sets

• Elimination of potential background from secondary antibody non-specific binding

• Compatibility with multi-color immunofluorescence experiments when combined with appropriate antibodies conjugated to spectrally distinct fluorophores

• Convenience for flow cytometry applications where direct detection improves sensitivity and reduces protocol complexity

What protocol optimizations are necessary for immunofluorescence applications with VPS4B Antibody, FITC conjugated?

For optimal immunofluorescence results:

• Fixation:

  • Test multiple fixation methods (4% paraformaldehyde, methanol, or acetone) to determine which best preserves VPS4B epitopes

  • Maintain consistent fixation times (typically 10-20 minutes at room temperature)

• Permeabilization:

  • Use gentle detergents (0.1-0.3% Triton X-100 or 0.1% saponin) to maintain subcellular structures

  • Optimize permeabilization time based on cell/tissue type (typically 5-15 minutes)

• Blocking:

  • Implement thorough blocking (5% normal serum, 3% BSA) for at least 30 minutes to reduce non-specific binding

  • Include 0.1% Tween-20 in blocking buffer to reduce background

• Antibody concentration:

  • Titrate antibody concentration starting from 1:50-1:200 dilutions

  • Incubate at 4°C overnight for optimal signal-to-noise ratio

• Controls:

  • Include isotype control (rabbit IgG-FITC)

  • Use tissues/cells with known VPS4B expression patterns as positive controls

How can researchers quantify VPS4B expression levels using VPS4B Antibody, FITC conjugated?

Quantification approaches include:

• Fluorescence microscopy quantification:

  • Capture images using identical exposure settings across all samples

  • Use automated image analysis software to define regions of interest (ROIs)

  • Measure mean fluorescence intensity within ROIs

  • Subtract background fluorescence from regions without cells/tissue

  • Normalize to cell number using nuclear counterstains

• Flow cytometry quantification:

  • Use standardized fluorescent beads to establish calibration curves

  • Report results as molecules of equivalent soluble fluorochrome (MESF)

  • Include appropriate positive and negative controls

  • Gate populations based on morphological parameters and viability markers

• Analysis considerations:

  • Assess both total expression and subcellular distribution patterns

  • Compare data across experimental conditions using appropriate statistical tests

  • Consider protein half-life and synthesis rates when interpreting results

What validation steps should be implemented to confirm VPS4B Antibody, FITC conjugated specificity?

Comprehensive validation should include:

• Western blot analysis:

  • Confirm single band of expected molecular weight (~44 kDa)

  • Compare results in samples with varying VPS4B expression levels

• Peptide competition assay:

  • Pre-incubate antibody with excess immunogenic peptide

  • Verify signal reduction in pre-absorbed samples compared to non-blocked antibody

• Knockout/knockdown confirmation:

  • Test antibody in VPS4B knockout or siRNA-treated samples

  • Verify signal reduction proportional to protein depletion

• Paralog specificity:

  • Test for cross-reactivity with recombinant VPS4A

  • Compare staining patterns with VPS4A-specific antibodies

  • Assess differential localization in cells expressing tagged VPS4A and VPS4B

How can the VPS4B Antibody, FITC conjugated be used to study ESCRT-dependent membrane remodeling?

To investigate ESCRT-dependent processes:

• Co-localization studies:

  • Perform dual immunofluorescence with other ESCRT components (CHMP4B, TSG101)

  • Use confocal or super-resolution microscopy to assess spatial relationships

  • Quantify co-localization using Pearson's correlation coefficient

• Temporal dynamics:

  • Implement live-cell imaging in cells expressing fluorescently-tagged ESCRT components

  • Compare fixed-cell timepoints using VPS4B Antibody, FITC conjugated

  • Quantify recruitment kinetics during ESCRT-mediated processes

• Functional studies:

  • Combine with expression of dominant-negative VPS4B mutants

  • Assess changes in VPS4B localization following ESCRT-III depletion

  • Correlate VPS4B recruitment with membrane scission events

• Comparative analysis:

  • Examine differential roles of VPS4A vs. VPS4B in specific processes

  • Investigate compensatory mechanisms following knockdown of either paralog

What approaches can be used to study VPS4B enzymatic activity in conjunction with antibody-based detection?

Integrated approaches include:

• Combined biochemical and immunological methods:

  • Use the Transcreener ADP2 Assay to measure VPS4B ATPase activity

  • Correlate activity measurements with expression levels detected by immunofluorescence

  • Optimize reaction conditions: 50 mM HEPES (pH 7.5), 10 mM MgCl₂, 50 mM KCl, 5 mM DTT, and 0.01% Triton at 37°C

• High-throughput screening:

  • Implement 96, 384, or 1536-well formats for inhibitor screening

  • Validate hits using immunofluorescence to assess effects on VPS4B localization

  • Leverage the robust Z' value (0.81) for reliable screening results

• Structure-function analysis:

  • Compare wild-type vs. mutant VPS4B for both enzymatic activity and localization

  • Assess ATP concentration effects (sub-Km concentration of 2 μM recommended)

  • Create standard curves using ATP/ADP to quantify reaction products

How do the functions of VPS4A and VPS4B differ, and how can researchers distinguish between them?

To differentiate between these paralogs:

• Functional distinctions:

  • VPS4A depletion causes more severe abscission delay than VPS4B depletion

  • VPS4A appears involved in earlier stages of abscission

  • Different phenotypic outcomes suggest non-redundant functions

• Experimental approaches to distinguish functions:

  • Use paralog-specific siRNAs/shRNAs to selectively deplete each protein

  • Perform rescue experiments with one paralog in cells depleted of the other

  • Compare localization patterns during different cellular processes (e.g., cytokinesis)

• Molecular tools:

  • Verify antibody specificity against recombinant VPS4A and VPS4B proteins

  • Use epitope-tagged constructs for selective visualization

  • Implement CRISPR/Cas9 to generate paralog-specific knockout cell lines

What are common issues with VPS4B Antibody, FITC conjugated and their solutions?

Frequent challenges and resolutions include:

• Weak or no signal:

  • Increase antibody concentration

  • Optimize antigen retrieval (test citrate buffer pH 6.0 vs. EDTA buffer pH 9.0)

  • Extend primary antibody incubation (overnight at 4°C)

  • Verify sample preparation (fixation time, permeabilization efficiency)

• High background:

  • Dilute antibody further

  • Extend washing steps (use at least 3 washes of 5-10 minutes each)

  • Implement additional blocking (normal serum matching secondary host)

  • Use more selective permeabilization conditions

• Photobleaching:

  • Minimize exposure to light during preparation

  • Use anti-fade mounting media

  • Capture images immediately after preparation

  • Consider using lower intensity excitation

• Inconsistent results:

  • Aliquot antibody to avoid repeated freeze-thaw cycles

  • Standardize protocols across experiments

  • Verify storage conditions (-20°C as recommended)

  • Check for lot-to-lot variation

What controls should be incorporated when designing experiments with VPS4B Antibody, FITC conjugated?

Essential controls include:

• Specificity controls:

  • Isotype control (rabbit IgG-FITC at same concentration)

  • Peptide competition/pre-absorption control

  • Knockdown/knockout samples (if available)

• Technical controls:

  • Unstained samples to assess autofluorescence

  • Single-color controls for spectral compensation in multi-color experiments

  • Secondary-only controls (for protocols using additional antibodies)

• Biological controls:

  • Tissues/cells with known VPS4B expression patterns

  • Samples treated with conditions known to alter VPS4B (e.g., ESCRT inhibition)

  • Comparison with alternative VPS4B antibodies

• Quantification controls:

  • Standard curves with known quantities of fluorophore

  • Calibration beads for flow cytometry applications

  • Internal reference standards for normalization

How should researchers optimize dual-staining protocols involving VPS4B Antibody, FITC conjugated?

For effective dual-staining:

• Fluorophore selection:

  • Choose secondary fluorophores with minimal spectral overlap with FITC (e.g., Cy3, Cy5)

  • Consider brightness differentials if targets have varying expression levels

  • Account for potential FRET if targets are in close proximity

• Protocol optimization:

  • Test sequential vs. simultaneous incubation of antibodies

  • Determine optimal fixation method compatible with all antibodies

  • Implement appropriate blocking to prevent cross-reactivity

• Imaging considerations:

  • Use narrow bandpass filters to minimize bleed-through

  • Perform sequential scanning for confocal microscopy

  • Consider spectral unmixing for closely overlapping fluorophores

• Controls for dual-staining:

  • Single-stained samples for each fluorophore

  • Concentration-matched isotype controls

  • Secondary-only controls to assess non-specific binding

How can VPS4B Antibody, FITC conjugated be used to study viral budding mechanisms?

For viral budding research:

• Visualization approaches:

  • Co-stain infected cells for VPS4B and viral proteins

  • Use time-course experiments to track VPS4B recruitment to budding sites

  • Implement super-resolution microscopy to visualize budding neck structures

• Functional analysis:

  • Compare VPS4B localization in cells infected with wild-type vs. late domain mutant viruses

  • Assess VPS4B recruitment following expression of dominant-negative ESCRT components

  • Quantify co-localization coefficients between VPS4B and viral structural proteins

• Mechanistic insights:

  • Combine with ATP-binding deficient VPS4B mutants

  • Compare recruitment patterns between different virus families

  • Correlate VPS4B activity with virion release efficiency

What approaches can be used to study VPS4B in cytokinetic abscission using the FITC-conjugated antibody?

To investigate cytokinetic functions:

• Temporal analysis:

  • Synchronize cells and immunostain at defined stages of cytokinesis

  • Co-stain with midbody markers (MKLP1, CEP55)

  • Quantify VPS4B recruitment kinetics during abscission

• Comparative studies:

  • Analyze differences in VPS4A vs. VPS4B recruitment to the midbody

  • VPS4A depletion causes more severe abscission delay than VPS4B

  • Assess potential functional redundancy through rescue experiments

• Mechanistic investigations:

  • Determine dependencies on ESCRT-III components for midbody recruitment

  • Analyze effects of ATPase-deficient mutants on abscission timing

  • Compare cancer vs. normal cells for potential differences in VPS4B function during cytokinesis

How can researchers effectively use VPS4B Antibody, FITC conjugated in exosome research?

For exosome applications:

• Characterization approaches:

  • Immunostain isolated exosomes for VPS4B presence

  • Perform immuno-electron microscopy to visualize VPS4B on exosomes

  • Compare VPS4B levels in exosomes from different cellular sources

• Mechanistic studies:

  • Track VPS4B association with multivesicular bodies during exosome biogenesis

  • Assess impact of VPS4B depletion on exosomal markers (CD63, syndecan)

  • Compare wild-type vs. ATPase-deficient VPS4B effects on exosome production

• Co-localization analysis:

  • Perform dual staining with established exosomal markers

  • Track temporal association during MVB maturation

  • Quantify degree of association under different physiological conditions