VCP Antibody, FITC conjugated

What cellular processes is VCP involved in and why is it an important research target?

VCP (p97/Transitional endoplasmic reticulum ATPase/TER ATPase) is a highly conserved AAA+ ATPase that plays vital roles in multiple cellular pathways. Research has demonstrated VCP's involvement in:

• Ubiquitin-proteasome system (UPS) and protein degradation pathways

• Endoplasmic reticulum-associated degradation (ERAD)

• DNA damage repair mechanisms

• Cell cycle regulation

• Viral replication processes

VCP is particularly important as a research target because it functions as a molecular segregase that extracts ubiquitinated proteins from cellular complexes and membranes for subsequent degradation . It has also emerged as a potential therapeutic target in cancer research, particularly in acute myeloid leukemia (AML), where VCP inhibition leads to unfolded protein response and apoptosis .

What are the primary applications for FITC-conjugated VCP antibodies in cellular research?

FITC-conjugated VCP antibodies serve several important research applications:

• Flow cytometry analysis of intracellular VCP expression and localization

• Monitoring VCP expression changes during disease progression or treatment

• Tracking VCP interactions with binding partners in live cells

• Studying VCP recruitment to specific cellular compartments during stress responses

These antibodies are particularly valuable for flow cytometry applications as demonstrated by validation data showing successful detection of VCP in human and mouse samples . For instance, flow cytometry analysis of HeLa cells using anti-VCP antibody conjugated to DyLight®488 (a fluorophore similar to FITC) demonstrated specific binding with clear separation from control samples .

How should researchers prepare samples for optimal FITC-VCP antibody staining in flow cytometry?

For optimal staining results when using FITC-conjugated VCP antibodies in flow cytometry:

• Cell fixation and permeabilization: Fix cells with 4% paraformaldehyde and permeabilize with an appropriate permeabilization buffer to facilitate intracellular staining, as VCP is predominantly an intracellular protein .

• Blocking: Block cells with 10% normal goat serum (or appropriate species serum matching secondary antibody) to reduce non-specific binding .

• Primary antibody incubation: Incubate cells with the FITC-conjugated VCP antibody at the recommended concentration (typically 1μg/1×10⁶ cells) for 30 minutes at 20°C .

• Controls: Include appropriate controls:

  • Isotype control antibody (e.g., FITC-conjugated rabbit IgG at the same concentration)

  • Unlabeled sample without primary or secondary antibody

  • Positive control samples with known VCP expression

• Analysis: Analyze samples using appropriate laser excitation (typically 488nm for FITC) and emission filter settings for the fluorochrome .

What are the critical factors affecting FITC-VCP antibody performance in immunofluorescence applications?

Several critical factors influence FITC-VCP antibody performance in immunofluorescence:

Research has shown that VCP antibody performance can vary significantly based on sample preparation. For instance, enzyme antigen retrieval was successfully employed for immunocytochemical detection of VCP in K562 cells, demonstrating the importance of optimizing antigen retrieval methods .

How can researchers validate the specificity of FITC-conjugated VCP antibody staining?

Validating antibody specificity is crucial for generating reliable scientific data. For FITC-conjugated VCP antibodies, researchers should:

• Knockdown/knockout controls: Use VCP-knockdown or knockout samples as negative controls to confirm antibody specificity. Published applications demonstrate the use of KD/KO approaches for validating VCP antibodies .

• Western blot confirmation: Perform western blot analysis using the same antibody (unconjugated version) to verify specificity by molecular weight (~89-100 kDa for VCP) .

• Immunoprecipitation validation: Conduct immunoprecipitation experiments to confirm antibody specificity. Positive IP detection of VCP has been reported in HeLa cells and mouse brain tissue .

• Blocking peptide competition: Pre-incubate the antibody with excess immunizing peptide to block specific binding sites.

• Multi-antibody comparison: Compare staining patterns with different antibodies targeting distinct VCP epitopes.

Cross-validation data indicates that anti-VCP antibodies have successfully detected the protein in multiple species (human, mouse, rat) and sample types, with observed molecular weights of 90-100 kDa, consistent with the predicted molecular weight of 89 kDa .

How can FITC-conjugated VCP antibodies be used to study VCP's role in viral replication mechanisms?

FITC-conjugated VCP antibodies offer valuable tools for investigating VCP's role in viral replication:

• Co-localization studies: Use FITC-VCP antibodies alongside viral protein markers to visualize recruitment of VCP to viral replication sites through confocal microscopy.

• Time-course analysis: Track VCP localization changes during viral infection progression using live-cell imaging or fixed timepoints.

• Immunoprecipitation followed by proteomics: Identify viral and host factors interacting with VCP during infection. Research has demonstrated that VCP binds to hantavirus glycoprotein Gn, suggesting VCP plays a critical role in hantavirus replication .

• Inhibitor studies: Monitor changes in VCP distribution following treatment with VCP inhibitors (e.g., NMS-873, CB-5083) to disrupt viral replication.

Studies have shown that VCP plays vital roles at multiple stages of hantavirus replication through interactions with viral glycoproteins, providing a potential target for antiviral intervention . The ability to visualize these interactions using fluorescently labeled antibodies allows researchers to better understand the spatiotemporal dynamics of these processes.

What considerations are important when using FITC-VCP antibodies to study unfolded protein response and proteostasis?

When investigating unfolded protein response (UPR) and proteostasis using FITC-VCP antibodies:

• UPR stimulus selection: Consider whether you're studying physiological or stress-induced UPR, as VCP functions differently under various stressors.

• Temporal considerations: Plan appropriate timepoints, as VCP's role in UPR evolves over time—early timepoints (1-4 hours) capture initial recruitment while later timepoints may reveal degradation processes.

• Co-staining strategy: Combine FITC-VCP antibodies with markers for:

  • ER stress (e.g., BiP/GRP78, CHOP, XBP1)

  • Ubiquitinated proteins (ubiquitin antibodies)

  • Proteasome components (e.g., PSMA3, PSMC3)

• Inhibitor controls: Include proteasome inhibitors (e.g., MG-132) and VCP inhibitors (e.g., CB-5083, NMS-873) as controls to distinguish VCP-dependent processes.

Research has shown that VCP inhibition induces robust accumulation of ubiquitylated protein species in a dose-dependent manner, mirroring effects of proteasome inhibition . Proteomics studies have identified diverse VCP substrates including components of the ubiquitin machinery, autophagy-related proteins, and DNA damage response factors .

How can dual-color flow cytometry with FITC-VCP antibodies be optimized for detecting apoptotic responses in cancer cells?

For dual-color flow cytometry detecting VCP and apoptotic markers:

• Panel design considerations:

  • Select apoptosis markers with non-overlapping emission spectra (e.g., PE-Annexin V with FITC-VCP)

  • Consider cell cycle markers (e.g., PI/RNase staining) for correlating VCP expression with cell cycle status

• Sequential staining protocol:
  a) First stain with surface markers (e.g., Annexin V) if applicable
  b) Fix and permeabilize cells for intracellular VCP staining
  c) Apply FITC-VCP antibody at optimal concentration (1μg/1×10⁶ cells)

• Compensation controls:

  • Single-stained samples for each fluorochrome

  • FMO (fluorescence minus one) controls

  • Unstained control

• Analysis strategy:

  • Gate on viable cells before VCP analysis

  • Create quadrant gates to correlate VCP expression with apoptotic status

  • Track VCP expression changes during apoptosis progression

This approach is supported by experimental protocols using PI/RNase staining buffer for cell cycle analysis and FITC Annexin V for apoptosis detection in conjunction with antibody staining . Research on AML cells treated with VCP inhibitors demonstrated that VCP inhibition leads to unfolded protein response and apoptosis, highlighting the importance of monitoring these processes simultaneously .

What methods can be used to investigate VCP interactions with ubiquitinated substrates using FITC-conjugated antibodies?

Several approaches can be employed to study VCP-substrate interactions:

• Co-immunoprecipitation followed by fluorescence detection:

  • Immunoprecipitate VCP using anti-VCP antibodies

  • Detect co-precipitated ubiquitinated proteins via western blotting

  • Research has demonstrated that VCP associates with both native and ubiquitin-conjugated forms of substrates like CSB

• Proximity ligation assay (PLA):

  • Use FITC-VCP antibody with anti-ubiquitin antibody

  • PLA signal indicates close proximity (<40nm) between VCP and ubiquitinated substrates

• FRET-based approaches:

  • Combine FITC-VCP antibody with red-fluorescent ubiquitin antibodies

  • FRET signal indicates direct molecular interaction

• Live-cell imaging:

  • Track dynamic recruitment of VCP to ubiquitinated substrates

  • Monitor substrate degradation kinetics

Studies using immunoprecipitation approaches have successfully demonstrated VCP's interaction with specific substrates and ubiquitinated proteins. For example, research showed that VCP/p97 segregase functions in ultraviolet radiation-induced ubiquitin-mediated CSB degradation, with both CSB and ubiquitinated CSB detected in VCP immunoprecipitates .

How can researchers differentiate between VCP's roles in ERAD versus other cellular processes using fluorescence microscopy?

To distinguish VCP's role in ERAD (Endoplasmic Reticulum-Associated Degradation) from other functions:

• Co-localization analysis with compartment-specific markers:

  • ER markers (e.g., calnexin, PDI)

  • Proteasome markers (e.g., 20S core)

  • P-body markers (as VCP has been implicated in P-body formation )

  • DNA repair foci markers (e.g., γH2AX)

• Temporal profiling:

  • Monitor VCP recruitment during ER stress induction

  • Track association with ERAD substrates over time

• Mutant VCP expression:

  • Compare localization of wild-type versus mutant VCP (e.g., ATPase-deficient mutants)

  • Research has utilized inducible Myc-WT or EQ-VCP/p97 systems for such comparisons

• Selective inhibitor approach:

  • Use ERAD-specific inhibitors versus general VCP inhibitors

  • Compare effects on VCP distribution and function

Research has demonstrated VCP's diverse functions across cellular compartments, including antigen entry into the endoplasmic reticulum critical for cross-presentation induced by certain vaccines . Studies have also shown VCP interactions with specific ERAD components and substrates, allowing researchers to track these associations using fluorescence-based approaches .

What are the most common technical issues with FITC-conjugated antibodies and how can they be resolved?

Technical validation data indicates that anti-VCP antibodies have been successfully used in multiple applications (WB, IHC, IF, IP) with species reactivity against human, mouse, and rat samples . Proper storage conditions (−20°C in PBS with 0.02% sodium azide and 50% glycerol, pH 7.3) help maintain antibody performance over time .

How can researchers validate that FITC conjugation has not altered the VCP antibody's epitope recognition properties?

To ensure FITC conjugation hasn't compromised antibody function:

• Comparative analysis:

  • Compare staining patterns between conjugated and unconjugated antibody versions

  • Verify that both detect the same ~89-100 kDa band in western blot

• Titration curve analysis:

  • Generate binding curves for both conjugated and unconjugated antibodies

  • Compare EC50 values to assess any affinity changes

• Competition assay:

  • Pre-incubate samples with excess unconjugated antibody before staining with FITC-conjugated antibody

  • Complete blocking indicates shared epitope recognition

• Cross-validation across applications:

  • Verify performance in multiple techniques (flow cytometry, IF, western blot)

  • Confirm consistent molecular weight detection (~90-100 kDa)

Research protocols indicate that FITC conjugation typically involves a 20-fold excess of FITC incubated with proteins in sodium bicarbonate/carbonate buffer (pH 9.5), with optimal FITC to protein molar ratios of approximately 2:3 . This controlled conjugation process helps maintain antibody specificity while providing the fluorescent tag necessary for detection.

How can FITC-VCP antibodies be utilized to investigate VCP's role in cancer progression and therapeutic resistance?

FITC-conjugated VCP antibodies offer valuable tools for cancer research:

• Tumor heterogeneity analysis:

  • Quantify VCP expression variability across tumor cell populations

  • Correlate with treatment response markers

  • Immunohistochemical analysis has demonstrated VCP expression in various cancer tissues including mammary cancer

• Treatment response monitoring:

  • Track changes in VCP localization and expression following therapy

  • Research has shown that prolonged treatment with VCP inhibitors like CB-5083 can lead to resistance mediated by mutations in VCP

• Drug combination studies:

  • Assess VCP expression/localization when combining VCP inhibitors with other therapeutics

  • Studies have demonstrated synergistic effects between VCP inhibitors and other drugs (cytarabine, venetoclax) in AML cell killing

• Patient-derived xenograft (PDX) analysis:

  • Compare VCP expression patterns between responsive and resistant tumors

  • Correlate with clinical outcomes

Recent research has highlighted VCP as a potential therapeutic target in AML, where VCP inhibition leads to lethal unfolded protein response . The ability to track VCP expression and localization changes using fluorescently labeled antibodies provides researchers with powerful tools to understand its role in cancer progression and therapeutic resistance.

What methodological approaches can be used to study VCP's involvement in viral replication using FITC-conjugated antibodies?

For investigating VCP's role in viral replication:

• Infection time-course analysis:

  • Track VCP redistribution at different stages of viral infection

  • Studies have shown VCP interacts with viral components like hantavirus glycoprotein Gn

• Super-resolution microscopy approaches:

  • Use techniques like STORM or STED with FITC-VCP antibodies to visualize nanoscale interactions with viral components

  • Combine with viral protein markers for co-localization analysis

• Fluorescence correlation spectroscopy (FCS):

  • Measure diffusion properties of VCP during infection

  • Detect complex formation with viral proteins

• FRAP (Fluorescence Recovery After Photobleaching):

  • Assess VCP mobility changes during viral infection

  • Compare dynamics in infected versus uninfected regions

Research has demonstrated that VCP plays vital roles in viral replication through interactions with viral glycoproteins. Specifically, immunoprecipitation and western blot analysis revealed that VCP binds to the hantavirus glycoprotein Gn, suggesting a critical interaction during viral replication . These findings highlight the importance of VCP as a potential target for antiviral intervention.

How can multiplexed approaches with FITC-VCP antibodies advance understanding of VCP in proteostasis networks?

Multiplexed analysis approaches offer comprehensive insights into VCP function:

• Multi-parameter flow cytometry:

  • Combine FITC-VCP with markers for:

    • Ubiquitinated proteins

    • Proteasome components

    • UPR sensors (BiP/GRP78, CHOP)

    • Cell cycle/apoptosis markers

• Mass cytometry (CyTOF) integration:

  • Convert FITC-VCP antibody to metal-tagged version

  • Simultaneously measure dozens of parameters to place VCP in broader proteostasis network

• Spatial proteomics approaches:

  • Combine FITC-VCP with organelle markers

  • Track VCP redistribution during proteotoxic stress

• Live-cell multiplexed imaging:

  • Monitor VCP dynamics alongside client proteins

  • Quantify temporal relationships between VCP recruitment and substrate degradation

Quantitative mass spectrometry-based proteomics has identified multiple VCP substrates, including ubiquitin machinery components, autophagy-related proteins, and DNA damage response factors . These findings provide a foundation for designing multiplexed approaches to further characterize VCP's role in coordinating these diverse cellular processes.