VPS29 Antibody, FITC conjugated

What is VPS29 and what are its primary functions relevant to antibody-based detection studies?

VPS29 is a critical component of the retromer complex, a conserved protein assembly that mediates endosomal protein sorting and trafficking. This protein plays essential roles in:

• Endocytic recycling of transmembrane proteins

• Regulation of synaptic transmission, particularly in aging adults

• Formation of stable protein complexes with VPS35 and VPS26

• Coordination with Rab7 and TBC1D5 to regulate retromer localization

Research in Drosophila has shown that VPS29 is dispensable for embryogenesis but required for retromer function in aging adults, including for synaptic transmission and survival . Unlike other retromer components like VPS35 and VPS26 whose mutations are lethal, VPS29 null mutations in Drosophila result in viable but shorter-lived animals, indicating its important role in maintaining cellular homeostasis over time .

What VPS29 isoforms exist and how might they affect antibody selection?

Research has identified three distinct human VPS29 isoforms with different amino-terminal sequences:

• VPS29A: The shortest form with the amino-terminal sequence 1MLVLVL6

• VPS29B: Contains four additional amino acids through alternative splicing, with the sequence 1MAGHRLVLVL10

• VPS29C: Features a significantly extended amino-terminal sequence of 28 amino acids

These isoforms exhibit differential binding properties with other proteins:

When selecting VPS29 antibodies, researchers must consider which isoform(s) they wish to detect, as antibodies raised against the core protein structure will recognize all isoforms, while those targeting N-terminal regions may be isoform-specific .

What cellular compartments should researchers expect to find VPS29 localization?

VPS29 predominantly localizes to endosomal structures as part of the retromer complex. Specific localization patterns include:

• Colocalization with early endosomal marker EEA1

• Association with endogenous VPS35-positive structures

• Cytosolic distribution when not assembled into retromer complexes

Immunofluorescence analysis of GFP-tagged VPS29 isoforms in H4 neuroglioma cells revealed that all three isoforms (VPS29A/B/C) retain association with endosomes where they colocalize with endogenous VPS35 and the early endosomal marker EEA1 . This endosomal localization is critical for VPS29's function in protein sorting and trafficking.

How should experiments be designed to distinguish between different VPS29 isoforms?

Distinguishing between VPS29 isoforms (A, B, and C) requires specific methodological approaches:

• Isoform-Specific Antibody Selection:

  • Use antibodies raised against the unique N-terminal sequences of each isoform

  • Validate antibody specificity against recombinant proteins of each isoform

• Molecular Biology Verification:

  • RT-PCR with isoform-specific primers targeting the junction regions

  • qPCR for quantitative assessment of isoform expression levels

  • Use of CRISPR-Cas9 to specifically tag or knockout individual isoforms

• Functional Characterization:

  • Immunoprecipitation followed by immunoblotting for specific interaction partners

  • VPS29C can be distinguished by its reduced binding to Retriever complex components and TBC1D5

  • VPS29B shows stronger TBC1D5 binding compared to VPS29A

A comprehensive approach would combine antibody-based detection with molecular validation and functional characterization to definitively identify specific isoforms in experimental systems.

How can researchers validate that their FITC-conjugated VPS29 antibody is detecting the correct target?

Multiple validation strategies should be employed to ensure specificity:

• Genetic Validation:

  • Testing antibodies on samples from VPS29 knockout models

  • Using CRISPR-Cas9 to generate VPS29-null cell lines as negative controls

  • Testing on samples with reduced VPS29 expression via RNA interference

• Biochemical Confirmation:

  • Western blotting to confirm detection of a single band of the expected molecular weight

  • Competitive binding assays with recombinant VPS29 protein

  • Immunoprecipitation followed by mass spectrometry to confirm target identity

• Localization Verification:

  • Co-localization with known VPS29 interactors (e.g., VPS35)

  • Signal reduction or disappearance in VPS29 knockout samples

  • Comparison with localization patterns of fluorescently tagged VPS29 constructs

Research described in the search results validated VPS29 antibody specificity by confirming the absence of protein detection in VPS29 null animals using western blotting .

What experimental controls are essential when studying VPS29 mutations or isoform-specific functions?

When investigating VPS29 mutations or isoform-specific functions, include these controls:

• Expression Level Controls:

  • Quantify expression levels of wild-type vs. mutant/isoform constructs

  • Use regulated expression systems to achieve comparable levels

  • Include western blot analysis to confirm protein expression

• Localization Controls:

  • Compare subcellular distribution patterns of wild-type and mutant/isoform proteins

  • Include co-localization with established markers (EEA1, VPS35)

  • Assess potential dominant-negative effects when overexpressing mutant forms

• Functional Rescue Controls:

  • Test the ability of each construct to rescue phenotypes in VPS29-deficient models

  • The L152E point mutation in VPS29 can serve as a functional control as it disrupts TBC1D5 binding

  • VPS29C constructs can be used to specifically assess Retromer-dependent but Retriever-independent functions

• Interaction Profile Controls:

  • Compare binding partners through immunoprecipitation followed by western blotting

  • Use quantitative proteomics to determine changes in the interactome

  • Include known differential binders (e.g., TBC1D5 binds strongly to VPS29B but weakly to VPS29C)

What fixation and permeabilization methods are optimal for VPS29 immunofluorescence studies?

Optimal fixation and permeabilization methods for VPS29 detection depend on the specific application:

• Fixation Protocols:

  • Standard: 4% paraformaldehyde for 10-15 minutes at room temperature

  • Epitope-sensitive: 2% paraformaldehyde with shorter fixation times (5-10 minutes)

  • Alternative: Methanol fixation (-20°C for 10 minutes) may better preserve some epitopes

  • Combined: Initial paraformaldehyde fixation followed by methanol permeabilization

• Permeabilization Methods:

  • Triton X-100: 0.1-0.2% for 5-10 minutes (standard)

  • Saponin: 0.1% for gentler permeabilization (may better preserve membrane structures)

  • Digitonin: 10-50 μg/ml for selective plasma membrane permeabilization

  • NP-40: 0.1% as an alternative to Triton X-100

• Cell Type Considerations:

  • Neuronal cells may require gentler fixation to preserve morphology

  • Epithelial cells often benefit from longer permeabilization times

  • Drosophila tissues may require specialized protocols as described in research using VPS29-GFP tagged constructs

Researchers should optimize these parameters for their specific cell type and antibody to achieve the best signal-to-noise ratio.

What are the best co-staining markers for VPS29 localization studies using FITC-conjugated antibodies?

For comprehensive VPS29 localization studies, consider these co-staining markers:

• Retromer Components:

  • VPS35: Core retromer component that directly interacts with VPS29

  • VPS26A/B: Core retromer components that assemble with VPS29

  • SNX1/2/5/6: Sorting nexins that associate with the retromer complex

• Endosomal Compartment Markers:

  • EEA1: Early endosome marker that co-localizes with VPS29

  • Rab7: Late endosome marker that shows altered levels in VPS29 mutants

  • Rab5: Early endosome marker for sorting stages

  • LAMP1: Lysosomal marker to distinguish from endosomal compartments

• Functional Interaction Partners:

  • TBC1D5: Interacts differently with VPS29 isoforms

  • Retriever components (VPS35L, VPS26C): Differential binding with VPS29 isoforms

  • Commander complex components (CCDC22, CCDC93): Interact with specific VPS29 isoforms

When using FITC-conjugated VPS29 antibodies, select compatible fluorophores for co-staining that minimize spectral overlap (such as Cy3, Cy5, or Alexa 647).

What sample preparation protocol differences are required when comparing VPS29 isoforms?

When comparing VPS29 isoforms, consider these protocol adjustments:

• Antibody Selection and Validation:

  • For pan-VPS29 detection: Use antibodies targeting conserved regions

  • For isoform-specific detection: Use antibodies against unique N-terminal sequences

  • Validate specificity using cells expressing single isoforms as controls

• Expression System Considerations:

  • For overexpression studies: Use C-terminal tags to preserve N-terminal differences

  • For endogenous detection: Use isoform-specific primers for RT-PCR verification

  • For CRISPR knock-in approaches: Insert tags that don't interfere with N-terminal regions

• Biochemical Analysis Adjustments:

  • For SDS-PAGE: Use gradient gels to resolve small size differences (VPS29A vs VPS29B)

  • For co-immunoprecipitation: Include detergent conditions that preserve complex integrity

  • For mass spectrometry: Consider enrichment strategies for less abundant isoforms

• Functional Assessments:

  • Exploit differential binding partners as readouts for specific isoforms

  • Include standard controls for each isoform (e.g., TBC1D5 binding as a readout for VPS29B)

  • Consider mutation of key residues (e.g., I15D in VPS29C) as functional controls

Research has successfully used these approaches to distinguish between VPS29 isoforms and their functional roles in retromer versus retriever assembly .

How can researchers address weak or non-specific signals when using FITC-conjugated VPS29 antibodies?

When encountering signal issues with FITC-conjugated VPS29 antibodies, implement these troubleshooting approaches:

• For Weak Signals:

  • Optimize antibody concentration through titration experiments

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

  • Implement signal amplification techniques (tyramide signal amplification)

  • Adjust fixation time to improve epitope accessibility

  • Implement antigen retrieval methods if appropriate for sample type

• For High Background:

  • Increase blocking time and concentration (5% BSA or normal serum)

  • Add additional wash steps with detergent-containing buffer

  • Pre-absorb antibodies with cell lysates from VPS29 knockout samples

  • Reduce antibody concentration and optimize incubation conditions

  • Use more stringent washing (higher salt concentration or detergent)

• For Non-specific Binding:

  • Validate antibody specificity against VPS29 knockout samples

  • Include additional blocking agents (e.g., normal serum from host species)

  • Use monovalent Fab fragments to block endogenous IgG in tissue samples

  • Filter antibody solutions before use to remove aggregates

  • Consider alternative antibody clones if available

• For Photobleaching Issues:

  • Use anti-fade mounting media specifically formulated for FITC

  • Minimize exposure time during imaging

  • Consider using newer generation dyes with better photostability if available

  • Reduce microscope lamp intensity and optimize imaging settings

What explains contradictory results when comparing VPS29 knockout phenotypes across different model systems?

Contradictory results in VPS29 knockout studies may arise from several factors:

• Species-Specific Differences:

  • Drosophila VPS29 knockout results in viable animals with reduced lifespan, unlike the embryonic lethality of VPS35/VPS26 mutants

  • In contrast, some mammalian studies show that VPS29 knockdown destabilizes the entire retromer complex

  • These differences highlight evolutionary variations in retromer complex stability requirements

• Isoform Compensation Mechanisms:

  • Different model systems may express varying levels of VPS29 isoforms

  • VPS29C retains strong Retromer binding while showing reduced Retriever association

  • Knockdown approaches targeting specific regions may affect isoforms differentially

• Technical Variations:

  • Complete knockout vs. knockdown approaches yield different results

  • RNA interference can have off-target effects not present in genetic knockouts

  • Different cell types may have varying requirements for VPS29 function

• Age-Dependent Phenotypes:

  • VPS29 null Drosophila show age-dependent defects in synaptic transmission

  • Embryonic vs. adult requirements for VPS29 differ significantly

  • Temporal aspects of experiments must be considered when comparing results

This complexity underscores the importance of comprehensive experimental design that accounts for isoform diversity, species differences, and temporal factors when studying VPS29 function.

How should researchers interpret unexpected changes in interaction partners when studying VPS29 mutations?

When unexpected changes in VPS29 interaction partners occur, consider these interpretation frameworks:

• Structural Impact Analysis:

  • The L152E mutation specifically disrupts TBC1D5 binding without affecting core retromer assembly

  • VPS29's hydrophobic groove mediates binding to multiple proteins including TBC1D5, ANKRD27, and FAM21

  • Mutations may disrupt shared binding interfaces affecting multiple partners

• Isoform-Specific Effects:

  • VPS29C shows dramatically reduced binding to Retriever components and TBC1D5 due to its N-terminal extension

  • The N-terminal extension in VPS29C creates an autoinhibitory mechanism

  • Mutations may have different effects depending on the isoform context

• Indirect vs. Direct Effects:

  • Primary binding partner changes may cause secondary effects on complex assembly

  • Changes in protein localization can alter the available interaction landscape

  • Regulatory modifications might be affected by mutations

• Data Validation Approaches:

  • Confirm unexpected results with multiple techniques (co-IP, proximity labeling, FRET)

  • Use structure-guided mutations to test binding interface hypotheses

  • Perform domain mapping to identify critical interaction regions

  • Compare in vitro binding with cellular context to identify potential cofactors

The research on VPS29 isoforms provides an excellent example where the unexpected reduction in VPS29C binding to Retriever components was explained through structural modeling and validated by targeted mutations (I15D) that restored binding .

How should quantitative co-localization of VPS29 with other markers be properly analyzed?

Proper quantitative co-localization analysis of VPS29 with other markers requires:

• Image Acquisition Standards:

  • Use confocal microscopy to minimize out-of-focus fluorescence

  • Acquire sequential scans to prevent bleed-through between channels

  • Maintain consistent acquisition parameters across comparable samples

  • Collect z-stacks to capture the full three-dimensional distribution

• Pre-Processing Steps:

  • Apply background subtraction using regions devoid of specific signal

  • Use deconvolution if appropriate to improve signal-to-noise ratio

  • Correct for chromatic aberration using multi-color beads as references

  • Apply threshold values consistently based on control samples

• Co-localization Coefficients:

  • Pearson's correlation coefficient: Measures linear correlation between signals

  • Manders' overlap coefficient: Reports fraction of pixels that co-localize

  • Object-based approaches: More appropriate for punctate endosomal structures

  • Distance-based analysis: For measuring proximity rather than overlap

• Statistical Analysis:

  • Compare coefficients across multiple cells (minimum 15-20 per condition)

  • Use appropriate statistical tests with corrections for multiple comparisons

  • Report both effect sizes and p-values

  • Consider spatial statistics approaches for clustered distributions

When analyzing VPS29 co-localization, researchers should focus on biologically relevant regions (e.g., endosomal compartments) rather than whole-cell measurements to achieve meaningful results.

What mathematical models best represent VPS29 complex assembly dynamics?

Mathematical modeling of VPS29 complex assembly can employ several approaches:

• Equilibrium Binding Models:

  • Simple binding equilibria for VPS29 associations with VPS35 and VPS26

  • Competition models for VPS29 incorporation into Retromer versus Retriever

  • Allosteric effects of the N-terminal extension in VPS29C on binding properties

  For example, the differential binding of VPS29 isoforms to Retromer versus Retriever can be modeled as:

  $\frac{[VPS29-Retromer]}{[VPS29-Retriever]} = \frac{K_{a,Retromer}}{K_{a,Retriever}}$

  Where the binding constants differ significantly between VPS29C and VPS29A/B .

• Kinetic Models:

  • Association and dissociation rate constants for complex formation

  • Multi-step assembly models incorporating sequential binding events

  • Temporal dynamics of complex formation and disassembly

• Spatial Models:

  • Reaction-diffusion equations for membrane recruitment dynamics

  • Compartmental models representing endosomal sorting functions

  • Spatial organization of Retromer versus Retriever complexes

• Network Models:

  • Protein interaction networks incorporating VPS29 and its binding partners

  • Dynamic remodeling of networks in response to mutations or isoform switching

  • Integration of multiple data types (proteomics, imaging, functional assays)

Research findings suggest that VPS29C exhibits an autoinhibitory mechanism through intramolecular interaction between its N-terminal extension and the hydrophobic groove , which could be modeled as:

$\frac{[VPS29C_{open}]}{[VPS29C_{closed}]} = K_{eq}$

Where the equilibrium constant determines the fraction of VPS29C available for interaction with partners requiring access to the hydrophobic groove.

How can researchers accurately quantify relative abundances of different VPS29 isoforms?

Accurate quantification of VPS29 isoform relative abundances requires:

• Transcriptome Analysis:

  • RNA-Seq with sufficient read depth to detect alternative splice junctions

  • Isoform-specific RT-qPCR with carefully designed primers spanning exon junctions

  • Targeted approaches like Nanostring to quantify specific isoform transcripts

  • Digital droplet PCR for absolute quantification of transcript numbers

• Proteome Analysis:

  • Mass spectrometry with isoform-specific peptide detection

  • Selected reaction monitoring (SRM) or parallel reaction monitoring (PRM) for targeted quantification

  • Use stable isotope-labeled peptide standards for absolute quantification

  • Western blotting with isoform-specific antibodies (when available)

• Calibration Approaches:

  • Include recombinant protein standards of each isoform at known concentrations

  • Use CRISPR-edited cell lines expressing single isoforms as references

  • Employ spike-in controls for normalization across samples

  • Account for extraction efficiency differences between isoforms

• Statistical Analysis:

  • Apply appropriate normalization methods for cross-sample comparisons

  • Use statistical models accounting for technical variation

  • Present data as relative abundances with confidence intervals

  • Calculate isoform ratios rather than absolute values when appropriate

Research on VPS29 isoforms has employed quantitative proteomics using TMT-based approaches to compare interaction partners, which can be adapted to quantify the isoforms themselves .