vcp Antibody, FITC conjugated

What is VCP and why is it important in cellular research?

VCP (Valosin Containing Protein) is a 220 kDa intra-acrosomal protein that plays critical roles in various cellular processes. It functions in ultraviolet radiation (UVR)-induced ubiquitin-mediated CSB degradation and participates in the ubiquitin-dependent sorting of membrane proteins to lysosomes . VCP also has roles in caveolin sorting in cells and indirectly regulates insulin-like growth factor receptor signaling pathways by controlling IGF1R receptor expression . Due to these diverse functions, VCP antibodies are valuable tools for investigating protein quality control, membrane trafficking, and stress response mechanisms.

What are the key specifications of commercially available FITC-conjugated VCP antibodies?

The most commonly used FITC-conjugated VCP antibody is a mouse monoclonal IgM antibody (clone Hs-14) that reacts with human and mouse samples . This antibody specifically recognizes VCP (valosin-containing protein), a 220 kDa intra-acrosomal protein . The antibody is validated for intracellular flow cytometry applications and has been cited in multiple research publications . The unconjugated version has also been validated for Western Blotting (WB), Immunocytochemistry (ICC), and Flow Cytometry (FACS) .

How specific is the VCP antibody, and what cross-reactivity should researchers be aware of?

The VCP antibody (clone Hs-14) demonstrates cross-reactivity with both human and mouse VCP proteins . The specificity has been verified through various applications, and the antibody has shown >95% purity by SDS-PAGE analysis . When selecting a VCP antibody for multi-species studies, researchers should note that while some VCP antibodies like the rabbit polyclonal (PA2137) can detect VCP in human, monkey, mouse, and rat samples , the FITC-conjugated mouse monoclonal (Hs-14) is validated specifically for human and mouse samples .

What is the optimal protocol for using FITC-conjugated VCP antibody in flow cytometry?

For intracellular flow cytometry using FITC-conjugated VCP antibody:

• Fix cells with 2-4% paraformaldehyde for 10-15 minutes at room temperature

• Permeabilize with 0.1% saponin or 0.1% Triton X-100 in PBS for 5-10 minutes

• Block with 2-5% normal serum from the same species as the secondary antibody

• Incubate with the FITC-conjugated VCP antibody (recommended dilution 1:50-1:200) for 30-60 minutes at room temperature or overnight at 4°C

• Wash cells 3 times with PBS containing 0.1% saponin

• Analyze by flow cytometry using 488nm excitation and appropriate emission filters for FITC detection

This protocol is optimized based on the validated applications for the FITC-conjugated Hs-14 clone, which is specifically suitable for intracellular flow cytometry with human and mouse samples .

How can researchers optimize immunoprecipitation experiments using VCP antibodies?

For effective immunoprecipitation of VCP and its binding partners:

• Prepare whole cell lysates in RIPA buffer (50 mM Tris-HCl pH 8.0, 150 mM NaCl, 1% Nonidet P-40, 0.5% deoxycholate, and protease inhibitors)

• Incubate lysates with anti-VCP antibody or control IgG at 4°C overnight

• Capture immunocomplexes using protein A Plus G-agarose beads

• For tagged VCP variants, use anti-Myc or anti-FLAG-agarose affinity gels for immunoprecipitation

• Analyze recovered proteins by Western blotting

This methodology has been successfully employed in studies investigating VCP interactions with CSB and ubiquitinated proteins . When studying VCP's role in protein degradation pathways, consider incorporating proteasome inhibitors or VCP/p97 inhibitors to accumulate ubiquitin conjugates for improved detection .

Can VCP antibodies be effectively used for immunohistochemistry applications, and what are the recommended tissue preparation protocols?

Yes, VCP antibodies can be effectively used for immunohistochemistry, though the FITC-conjugated version is primarily optimized for flow cytometry. For IHC applications:

• For paraffin-embedded sections: Use heat-mediated antigen retrieval in citrate buffer (pH 6.0) for 20 minutes

• Block with 10% normal serum from the same species as the secondary antibody

• Incubate with primary VCP antibody (1 μg/ml) overnight at 4°C

• For detection, use a biotinylated secondary antibody followed by Streptavidin-Biotin-Complex with DAB as chromogen

This protocol has been validated with rabbit anti-VCP antibody on human mammary cancer tissue and rat cerebellum tissue . While FITC-conjugated antibodies are not typically used for IHC with DAB detection, if fluorescence detection is desired, the sections can be mounted with an anti-fade mounting medium after primary antibody incubation and visualized using a fluorescence microscope with appropriate filters.

How can researchers distinguish between different functional pools of VCP using antibody-based techniques?

To distinguish between different functional pools of VCP:

• Subcellular Fractionation Approach:

  • Separate nuclear, cytoplasmic, membrane, and chromatin fractions

  • Perform Western blotting with VCP antibodies on each fraction

  • Compare distribution patterns under different cellular conditions

• Co-immunoprecipitation Strategy:

  • Use VCP antibodies to pull down protein complexes

  • Identify different interacting partners by mass spectrometry or Western blotting

  • Correlate interactors with specific VCP functions

• Double/Triple Immunostaining:

  • Combine FITC-conjugated VCP antibody with antibodies against known VCP interactors

  • Use confocal microscopy to assess colocalization patterns

  • Quantify colocalization using appropriate software (e.g., ImageJ with colocalization plugins)

This multi-faceted approach can help researchers distinguish between VCP involved in protein degradation, membrane trafficking, or DNA damage response pathways .

What are the key considerations when using VCP antibodies to study ubiquitin-dependent protein degradation pathways?

When studying VCP's role in ubiquitin-dependent protein degradation:

• Experimental Design Considerations:

  • Include appropriate controls: proteasome inhibitors (MG132) and VCP/p97 inhibitors

  • Compare ubiquitination patterns with and without inhibitors

  • Use denaturing conditions during immunoprecipitation to eliminate non-covalent interactions

• Technical Approach:

  • Immunoprecipitate VCP to detect associated ubiquitinated proteins

  • Reciprocally, immunoprecipitate ubiquitinated proteins to detect VCP association

  • Use antibodies specific for different ubiquitin linkages (K48, K63) to distinguish degradation from signaling

• Data Interpretation:

  • An increase in ubiquitinated proteins after VCP inhibition suggests VCP's role in degradation

  • Co-immunoprecipitation of VCP with ubiquitinated proteins confirms direct involvement

These approaches have been used to demonstrate that both proteasome and VCP/p97 inhibition allows accumulation of ubiquitin conjugates, and that VCP/p97 protein associates with CSB and ubiquitinated CSB .

How do researchers troubleshoot non-specific binding or high background when using FITC-conjugated VCP antibodies?

To minimize non-specific binding and background with FITC-conjugated VCP antibodies:

• Optimizing Fixation/Permeabilization:

  • Test different fixatives (paraformaldehyde vs. methanol)

  • Adjust permeabilization conditions (concentration and incubation time)

  • Include BSA or serum in all buffers to block non-specific sites

• Antibody Dilution and Incubation:

  • Perform titration experiments to determine optimal antibody concentration

  • Compare different incubation temperatures and durations

  • Include appropriate isotype control (mouse IgM-FITC) at the same concentration

• Reducing Autofluorescence:

  • For tissue samples, treat with 0.1% Sudan Black in 70% ethanol

  • For cells with high autofluorescence, use quenching agents like 0.1% sodium borohydride

  • Select appropriate filters to minimize spectral overlap

• Advanced Solutions:

  • If persistent background occurs, consider indirect detection with unconjugated primary and FITC-conjugated secondary antibody

  • Implement additional blocking steps with normal serum and/or commercial blocking reagents

These approaches help ensure specific detection of VCP protein while minimizing artifacts that could complicate data interpretation.

How do different VCP antibody clones compare in terms of epitope recognition and functional studies?

Various VCP antibody clones recognize different epitopes and offer complementary research applications:

This diversity of antibodies allows researchers to target specific domains of VCP for different experimental questions. For studying protein interactions, C-terminal antibodies may be preferable, while N-terminal antibodies might be better for detecting full-length protein.

What are the implications of studying VCP in different cellular compartments using FITC-conjugated antibodies?

VCP functions in multiple cellular compartments, and FITC-conjugated antibodies can help reveal compartment-specific roles:

• Nuclear Functions:

  • VCP participates in DNA damage repair and chromatin remodeling

  • FITC-conjugated antibodies can visualize nuclear translocation following stress

  • Co-staining with nuclear markers helps quantify nuclear/cytoplasmic ratios

• Cytoplasmic Functions:

  • VCP mediates protein degradation and organelle quality control

  • FITC-labeled antibodies can track cytoplasmic aggregates or structures

  • Particularly useful for studying stress granules or aggresome formation

• Membrane-Associated Functions:

  • VCP plays roles in membrane protein trafficking and ERAD (ER-associated degradation)

  • FITC-conjugated antibodies can visualize ER/Golgi localization

  • Co-staining with organelle markers enables precise localization

• Methodological Considerations:

  • Optimize fixation/permeabilization for each compartment

  • Use compartment-specific markers for co-localization studies

  • Consider confocal microscopy for precise spatial resolution

This compartment-specific analysis helps researchers dissect the multifunctional nature of VCP in normal physiology and disease states.

How can researchers effectively use VCP antibodies to study neurodegenerative diseases?

VCP mutations are associated with several neurodegenerative disorders, and antibody-based approaches offer valuable insights:

• Disease Model Systems:

  • In patient-derived cells, VCP antibodies can detect altered localization or aggregation

  • In transgenic mouse models, immunohistochemistry can reveal pathological changes

  • In cerebellum tissue, VCP antibodies have been validated for detecting expression patterns

• Methodological Approach:

  • For cerebellar sections, use heat-mediated antigen retrieval in citrate buffer

  • Block with 10% goat serum and incubate with 1μg/ml VCP antibody overnight

  • For fluorescent detection, use FITC-conjugated antibodies directly or as secondary detection

  • For chromogenic detection, use biotinylated secondary antibodies with DAB development

• Research Applications:

  • Detection of VCP aggregates in disease models

  • Analysis of VCP interaction with disease-associated proteins

  • Assessment of VCP distribution in affected versus unaffected brain regions

• Advanced Analysis:

  • Quantitative image analysis to measure VCP expression levels

  • Co-localization studies with ubiquitin or TDP-43 to assess pathological associations

  • Correlation of VCP patterns with disease progression markers

These approaches help elucidate VCP's role in neurodegeneration and may identify potential therapeutic targets.

How can new technologies enhance the utility of VCP antibodies in protein degradation research?

Emerging technologies are expanding the applications of VCP antibodies:

• Super-Resolution Microscopy:

  • FITC-conjugated VCP antibodies can be used with STORM or PALM microscopy

  • Enables visualization of VCP-containing complexes at nanometer resolution

  • Reveals spatial organization of degradation complexes not visible with conventional microscopy

• Proximity Labeling Techniques:

  • Combining VCP antibodies with BioID or APEX2 approaches

  • Identifies proteins in close proximity to VCP in living cells

  • Provides dynamic information about VCP interaction networks

• Live-Cell Imaging:

  • While FITC-conjugated antibodies are typically used in fixed cells, newer cell-permeable nanobodies

  • May enable tracking of VCP movements in live cells

  • Can be combined with optogenetic tools to manipulate VCP function

• Single-Cell Analysis:

  • FITC-conjugated VCP antibodies enable flow cytometry-based single-cell analysis

  • Can be combined with other markers for multi-parameter analysis

  • Allows correlation of VCP expression with cellular states or disease biomarkers

These technological advances will help researchers gain more detailed insights into VCP's roles in protein homeostasis and degradation pathways.

What are the key methodological considerations when studying VCP in the context of cellular stress responses?

When investigating VCP's role in stress responses:

• Experimental Design:

  • Include time-course analyses to capture dynamic changes in VCP localization

  • Compare multiple stress inducers (UVR, proteasome inhibitors, oxidative stress)

  • Include both acute and chronic stress conditions

• Technical Approach:

  • For UVR experiments, follow protocols similar to those studying CSB degradation

  • Use both Western blotting and flow cytometry to quantify changes

  • Consider cell fractionation to detect stress-induced translocation

• Controls and Validation:

  • Include VCP inhibitors to confirm specific involvement

  • Use siRNA/shRNA knockdown as complementary approach

  • Compare results across multiple cell types to ensure generalizability

• Advanced Analysis:

  • Correlate VCP patterns with markers of different stress responses

  • Quantify changes in VCP-substrate interactions during stress

  • Analyze post-translational modifications of VCP using specialized antibodies

These methodological considerations ensure robust data when studying VCP's dynamic roles in cellular stress response pathways.

How can researchers integrate VCP antibody-based studies with ubiquitin chain analysis?

To comprehensively study VCP's role in ubiquitin-mediated processes:

• Integrated Approach:

  • Use VCP antibodies to immunoprecipitate VCP-containing complexes

  • Analyze associated ubiquitin chains using linkage-specific antibodies

  • Compare patterns before and after VCP inhibition

• Technical Workflow:

  • First step: Immunoprecipitate with VCP antibody

  • Second step: Probe with antibodies against different ubiquitin linkages (K48, K63, etc.)

  • Alternative approach: Use ubiquitin linkage antibodies for immunoprecipitation, then probe for VCP

• Data Interpretation Framework:

  • K48-linked chains typically signal for proteasomal degradation

  • K63-linked chains often indicate non-degradative functions

  • Changes in linkage patterns after VCP manipulation suggest specific roles

• Validation Strategy:

  • Use ubiquitin mutants to confirm linkage specificity

  • Apply mass spectrometry to identify exact ubiquitination sites

  • Correlate findings with functional outcomes (protein degradation, complex formation)

This integrated approach has revealed that VCP/p97 segregase functions in UVR-induced ubiquitin-mediated CSB degradation, with both proteasome and VCP/p97 inhibition allowing accumulation of ubiquitin conjugates .