VCP Antibody, HRP conjugated

What is VCP and why is it an important research target?

VCP (valosin-containing protein) is a highly conserved 89 kDa protein (observed molecular weight 90-100 kDa) that serves multiple critical cellular functions. It belongs to the AAA+ (ATPases Associated with diverse cellular Activities) family and plays essential roles in protein quality control, membrane fusion, DNA damage repair, and cell cycle regulation. VCP's involvement in numerous neurodegenerative disorders, cancer progression, and viral infections makes it a significant target for research across multiple disciplines . The protein contains four short consensus repeat (SCR) domains that are crucial for its various functional interactions, particularly in contexts like viral immune evasion mechanisms where VCP can inhibit complement activation through interactions with C3b/C4b .

What are the primary applications for HRP-conjugated VCP antibodies?

HRP-conjugated VCP antibodies serve as powerful tools for direct detection of VCP in multiple experimental contexts without requiring secondary antibody incubation. The primary applications include:

• Western blotting with enhanced sensitivity and reduced background

• Immunohistochemistry (IHC) with simplified protocols and cleaner signal

• ELISA assays with direct detection capabilities

• Immunocytochemistry for subcellular localization studies

• Flow cytometry applications requiring direct detection

HRP conjugation provides significant advantages in experimental workflows where minimizing cross-reactivity or reducing total incubation steps is desirable .

How does VCP antibody reactivity vary across species?

VCP antibodies demonstrate varying degrees of cross-reactivity across species due to the highly conserved nature of the protein. Based on extensive validation data:

When designing experiments involving non-human samples, it is crucial to verify specific epitope conservation. Most commercially available VCP antibodies are raised against human VCP fusion proteins, with cross-reactivity to rodent models being particularly valuable for translational research . The conservation across mammalian species facilitates comparative studies while requiring careful validation when extending to more evolutionarily distant organisms.

How should I determine the optimal dilution of HRP-conjugated VCP antibody for my experiment?

Determining the optimal dilution for HRP-conjugated VCP antibody requires systematic titration specific to each experimental application. For unconjugated VCP antibodies, recommended dilutions typically range from 1:2000-1:16000 for Western blot and 1:100-1:400 for IHC . For HRP-conjugated versions, start with a higher dilution (approximately 2-5× more dilute than recommended for unconjugated antibody) and perform a systematic optimization:

• Prepare a dilution series (e.g., 1:5000, 1:10000, 1:20000, 1:40000 for Western blot)

• Test against both positive controls and experimental samples

• Evaluate signal-to-noise ratio, not just signal intensity

• Include appropriate negative controls (isotype control and secondary-only controls)

• Verify specificity through knockout/knockdown validation when possible

Optimal dilution is highly dependent on sample type, protein abundance, and detection method sensitivity. The ideal working dilution achieves clear specific signal with minimal background .

How can I distinguish between specific and non-specific binding in my VCP antibody experiments?

Distinguishing specific from non-specific binding requires multiple validation approaches:

• Knockout/Knockdown Validation: The gold standard approach involves comparing signal between wild-type and VCP-depleted samples. Published literature demonstrates successful validation of VCP antibodies through RNAi experiments targeting VCP .

• Peptide Competition: Pre-incubate antibody with excess VCP recombinant protein or immunogenic peptide before application to sample.

• Molecular Weight Verification: Confirm signal corresponds to expected molecular weight (89-100 kDa for VCP) with appropriate size markers.

• Multiple Antibody Verification: Compare staining patterns using antibodies targeting different VCP epitopes.

• Cross-Species Validation: Verify consistent detection pattern across species with known VCP conservation.

For HRP-conjugated antibodies specifically, include enzyme-only controls to exclude potential direct HRP interactions with endogenous peroxidases or sample components .

What protein extraction methods are optimal for preserving VCP epitopes?

VCP is primarily located in the cytoplasm but can translocate to the nucleus, ER, and other organelles under various cellular conditions. Optimal extraction methods should preserve epitope integrity while effectively solubilizing VCP from all cellular compartments:

• Standard RIPA Buffer: Effective for most applications, containing:

  • 150 mM NaCl

  • 1.0% NP-40 or Triton X-100

  • 0.5% sodium deoxycholate

  • 0.1% SDS

  • 50 mM Tris-HCl pH 8.0

  • Protease inhibitor cocktail

• Gentle Non-ionic Detergent: For co-immunoprecipitation studies preserving protein-protein interactions:

  • 150 mM NaCl

  • 1% NP-40 or 0.5% Triton X-100

  • 50 mM Tris-HCl pH 7.5

  • Protease and phosphatase inhibitors

• Subcellular Fractionation: For studies investigating VCP localization, sequential extraction of cytoplasmic, membrane, nuclear, and chromatin-bound fractions yields more detailed insights.

Temperature control during extraction (4°C) and rapid processing are critical for preserving epitope integrity and preventing artifactual post-lysis modifications .

How do I properly validate specificity of HRP-conjugated VCP antibodies in different experimental systems?

Comprehensive validation of HRP-conjugated VCP antibodies requires a multi-tiered approach that addresses both antibody specificity and HRP functionality:

• Epitope-specific Validation:

  • Western blot comparison against recombinant VCP protein

  • Immunoprecipitation followed by mass spectrometry verification

  • Side-by-side comparison with well-validated unconjugated antibodies

  • Detection using knockout/knockdown systems as demonstrated in published studies

• HRP Activity Verification:

  • Substrate conversion kinetics assessment

  • Temperature and pH stability profiling

  • Interference testing with sample-specific inhibitors

  • Signal linearity assessment across concentration ranges

• Application-specific Controls:

  • For Western blotting: stripped and reprobed membranes

  • For IHC: isotype controls and absorption controls

  • For ELISA: standard curve verification with recombinant protein

The validation strategy should be tailored to the specific research question and experimental system. Documented validation in publications adds additional confidence in antibody performance .

What experimental approaches can resolve contradictory results between VCP protein levels and function?

Resolving contradictions between VCP protein abundance and functional readouts requires sophisticated experimental design addressing multiple levels of VCP regulation:

• Post-translational Modification Analysis:

  • Assess VCP phosphorylation, ubiquitination, or SUMOylation status using modification-specific antibodies

  • Employ 2D-gel electrophoresis to separate VCP isoforms

  • Use mass spectrometry to identify and quantify specific modifications

• Complex Formation Assessment:

  • Analyze VCP interactions with cofactors (e.g., Ufd1-Npl4, p47)

  • Perform blue native PAGE to preserve native protein complexes

  • Use proximity ligation assays to detect specific interaction partners in situ

• Subcellular Localization Studies:

  • Employ fractionation followed by immunoblotting

  • Use immunofluorescence with organelle co-markers

  • Perform immunoelectron microscopy for high-resolution localization

• Activity-based Probes:

  • Utilize ATPase activity assays to measure VCP enzymatic function

  • Apply FRET-based biosensors to monitor VCP conformational changes

  • Develop client protein degradation assays as functional readouts

How can I design experiments to study VCP's role in specific protein degradation pathways?

Designing experiments to investigate VCP's involvement in protein degradation pathways requires careful attention to pathway-specific factors and readouts:

• ERAD Pathway Analysis:

  • Utilize model ERAD substrates (e.g., αTCR, α1-antitrypsin variants) alongside VCP manipulation

  • Monitor substrate retrotranslocation using protease protection assays

  • Assess ER chaperone associations (BiP, Grp94) through co-immunoprecipitation

• Autophagy Connection:

  • Perform dual monitoring of autophagic flux markers (LC3-II, p62) with VCP modulation

  • Use tandem fluorescent-tagged autophagy substrates to distinguish autophagosome formation from fusion

  • Analyze VCP co-localization with autophagy components under basal and induced conditions

• Proteasomal Degradation Assessment:

  • Compare effects of VCP inhibition versus proteasome inhibition on substrate accumulation

  • Monitor polyubiquitinated protein profiles following VCP manipulation

  • Employ fluorescent proteasome substrates with varying degron sequences

• Selective Substrate Analysis:

  • Develop pulse-chase experiments with pathway-specific substrates

  • Utilize proximity labeling techniques to identify VCP-associated substrates

  • Compare degradation kinetics across multiple VCP-dependent substrates

Research has demonstrated that VCP depletion affects different substrates with varying degrees of severity, suggesting pathway-specific roles that require careful experimental design to elucidate .

Why might I observe multiple bands when using VCP antibodies in Western blotting?

The appearance of multiple bands in VCP Western blots can result from several biological and technical factors:

• Post-translational Modifications:

  • Phosphorylation can cause mobility shifts (observed molecular weight of VCP is 90-100 kDa versus calculated 89 kDa)

  • Ubiquitination generates higher molecular weight species

  • Other modifications (SUMOylation, acetylation) may alter migration

• Proteolytic Processing:

  • Stress-induced cleavage by caspases or other proteases

  • Sample preparation artifacts (insufficient protease inhibition)

  • Cell-type specific processing patterns

• Splice Variants:

  • Alternative splicing producing different VCP isoforms

  • Tissue-specific expression patterns of variants

• Technical Factors:

  • Incomplete denaturation of VCP complexes

  • Protein overloading causing smearing

  • Non-specific antibody binding to related AAA+ family proteins

To distinguish between these possibilities:

• Compare patterns across different cell/tissue types

• Test with antibodies recognizing different VCP epitopes

• Perform peptide competition assays to confirm specificity

• Verify through immunoprecipitation followed by mass spectrometry analysis

How can I optimize VCP antibody performance in formalin-fixed paraffin-embedded (FFPE) tissues?

Optimizing HRP-conjugated VCP antibody performance in FFPE tissues requires systematic antigen retrieval optimization and signal enhancement:

• Antigen Retrieval Optimization:

  • Compare heat-induced epitope retrieval methods:

    • Citrate buffer (pH 6.0)

    • TE buffer (pH 9.0) - often preferred for VCP detection

    • EDTA buffer (pH 8.0)

  • Test retrieval duration and temperature variations

  • Consider enzymatic retrieval for challenging samples

• Signal Enhancement Strategies:

  • Implement avidin-biotin blocking for high-background tissues

  • Utilize tyramide signal amplification for low-abundance detection

  • Optimize endogenous peroxidase quenching (3% H₂O₂, 10 minutes)

  • Test alternative blocking agents (animal-free blockers for reduced background)

• Protocol Modifications:

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

  • Optimize antibody dilution specifically for FFPE sections

  • Consider addition of penetration enhancers for improved antibody access

• Validation Methods:

  • Include known positive control tissues (brain, colon, lung cancer tissues have demonstrated positive IHC signals)

  • Implement absorption controls with immunizing peptide

  • Compare with fresh frozen tissue immunostaining patterns

Research has demonstrated successful VCP immunodetection in human gliomas, colon cancer, and lung cancer tissues with proper optimization of antigen retrieval conditions .

What approaches can resolve high background issues with HRP-conjugated VCP antibodies?

High background with HRP-conjugated VCP antibodies can be systematically addressed through multiple optimization strategies:

• Antibody-specific Optimizations:

  • Increase dilution factor (often 2-5× higher than unconjugated antibodies)

  • Reduce incubation time or temperature

  • Filter antibody solution through 0.22 μm filter before use

  • Consider using carrier proteins (BSA, casein) in diluent

• Blocking Optimizations:

  • Test alternative blocking agents (milk, BSA, commercially available blockers)

  • Extend blocking duration (2-4 hours or overnight)

  • Include mild detergents in washing and blocking buffers (0.05-0.1% Tween-20)

  • Add serum matching secondary host species (for comparison with unconjugated systems)

• Substrate-related Optimizations:

  • Reduce substrate incubation time

  • Test alternative HRP substrates with different sensitivity/background profiles

  • Include HRP inhibitors in blocking step

  • Ensure substrates are fresh and properly stored

• Sample-specific Approaches:

  • Increase washing duration and number of wash steps

  • Pre-absorb antibodies with proteins from the experimental system

  • Block endogenous biotin for avidin-biotin detection systems

  • For tissues, implement dual peroxidase/phosphatase blocking

Systematic optimization is essential, with documented evidence showing significant improvements in signal-to-noise ratio when appropriate blocking and washing conditions are implemented .

How can I design experiments to investigate the role of VCP in viral mechanisms of immune evasion?

Investigating VCP's role in viral immune evasion requires sophisticated experimental approaches that combine virology and immunology techniques:

• Complement Interaction Studies:

  • Analyze VCP binding to C3b/C4b using antibodies targeting different SCR domains

  • Employ monoclonal antibodies that inhibit VCP's complement control activity (e.g., MAbs 2E5, 3D1, and 3F11)

  • Develop complement neutralization assays measuring VCP's ability to abolish complement-enhanced virus neutralization

• Domain-specific Functional Analysis:

  • Generate and express VCP constructs with mutations in specific SCR domains

  • Utilize domain-blocking antibodies to assess function of individual domains

  • Perform competitive binding assays with domain-specific antibodies

• Viral Replication Impact:

  • Compare wildtype virus with VCP-deficient or VCP-mutant viruses

  • Assess viral replication in the presence of domain-specific blocking antibodies

  • Measure complement activation in the presence/absence of functional VCP

• Translation to Therapeutic Approaches:

  • Screen for small molecule inhibitors of VCP-complement interactions

  • Test therapeutic potential of anti-VCP monoclonal antibodies

  • Develop vaccination strategies targeting viral VCP

Research has established that structural elements involved in VCP's interaction with C3b/C4b are located within SCR domains 2, 3, and 4, providing a foundation for targeted experimental designs .

What methods are most effective for studying VCP's role in protein quality control pathways across different disease models?

Studying VCP in protein quality control across disease models requires integrating multiple experimental systems:

• Cellular Stress Response Analysis:

  • Compare VCP recruitment to aggregates under different stressors (proteasome inhibition, ER stress)

  • Analyze co-localization with different quality control markers (polyubiquitin, chaperones)

  • Monitor formation of stress-induced compartments following VCP manipulation

• Client Protein Degradation Kinetics:

  • Track degradation of model substrates (e.g., αTCR, α1-antitrypsin variants) in disease backgrounds

  • Compare effects of VCP inhibition versus conditional knockdown/knockout

  • Utilize fluorescent timers to monitor substrate stability in real-time

• Disease-relevant Model Systems:

  • Generate cell models expressing disease-associated VCP mutations

  • Develop patient-derived iPSC models with VCP mutations

  • Utilize conditional tissue-specific VCP knockout animal models

• Multi-omics Integration:

  • Perform proteomics to identify VCP interactors under disease conditions

  • Utilize proximity labeling to capture transient VCP interactions

  • Integrate transcriptomics to identify compensatory mechanisms

Evidence demonstrates that VCP depletion affects different substrates with varying severity, indicating pathway-specific roles that may explain disease phenotype variability. For instance, RNAi of VCP was shown to stabilize cytosolic substrates while having minimal effects on ERAD substrates like δCD3 and α1-antitrypsin .

How can epitope mapping of VCP antibodies inform functional studies of VCP complex assembly?

Epitope mapping of VCP antibodies provides powerful insights into protein complex assembly and function:

• Structure-Function Relationship Analysis:

  • Map antibody epitopes to specific functional domains of VCP

  • Correlate epitope accessibility with VCP conformational states

  • Utilize antibodies recognizing different epitopes to probe complex assembly states

• Advanced Mapping Techniques:

  • Employ fragment-based mapping using truncated VCP constructs

  • Perform competition assays between different monoclonal antibodies

  • Utilize hydrogen-deuterium exchange mass spectrometry with and without antibody binding

• Functional Impact Assessment:

  • Compare effects of antibodies binding different epitopes on VCP ATPase activity

  • Assess impact on co-factor recruitment (Ufd1-Npl4, p47)

  • Analyze changes in substrate processing with epitope-specific antibodies

• Application to Complex Assembly Studies:

  • Use epitope-mapped antibodies as probes for complex assembly states

  • Develop conformation-specific antibodies based on epitope mapping data

  • Create biosensor systems incorporating epitope-specific antibody fragments

Research has demonstrated successful epitope mapping of VCP antibodies, revealing that antibodies targeting different SCR domains have distinct functional effects. For example, antibodies recognizing SCR domains 2, 3, and 4 blocked VCP's complement control activity, while antibodies targeting SCR 1 did not interfere with function .