vimp Antibody

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

VIMP (VCP-interacting membrane protein), also known as Selenoprotein S or SELENOS, is a transmembrane protein involved in key cellular processes. VIMP is significant in research due to its role in endoplasmic reticulum (ER) function, including participation in ER-associated degradation (ERAD) and its interactions with cytoskeletal elements. Studies have demonstrated that VIMP interacts with CLIMP-63, suggesting its involvement in ER-microtubule bundling, which has implications for cellular architecture and function . VIMP's participation in multiple cellular pathways makes it a valuable target for investigating ER stress responses, selenoprotein biology, and potential roles in disease states including cancer and inflammatory conditions.

What types of VIMP antibodies are available for research?

Current research-grade VIMP antibodies include polyclonal antibodies raised in rabbits using synthetic peptide immunogens derived from internal residues of human VCP-interacting membrane protein . These antibodies are predominantly unconjugated and require appropriate secondary antibodies for detection. Available VIMP antibodies have been validated primarily for immunohistochemistry (IHC) applications on human tissue samples, with confirmation of specificity for endogenous levels of total VIMP protein . When selecting a VIMP antibody, researchers should consider factors such as species reactivity (primarily human for current offerings), application compatibility, clonality (polyclonal being most common), and validation data availability.

How can I verify the specificity of a VIMP antibody?

Verification of VIMP antibody specificity involves multiple complementary approaches. First, examine existing validation data from manufacturers, including immunohistochemical analyses that demonstrate specific staining patterns consistent with known VIMP localization . For independent validation, researchers should consider:

• Western blot analysis using positive control samples (tissues known to express VIMP) alongside negative controls

• Immunoprecipitation followed by mass spectrometry to confirm target pull-down

• Antibody performance in VIMP-knockout or knockdown systems

• Co-immunoprecipitation studies to confirm expected protein-protein interactions, such as VIMP-CLIMP-63 interaction

A thorough approach combines multiple validation techniques to establish confidence in antibody specificity before proceeding with critical experiments.

What tissue types show notable VIMP expression patterns?

VIMP expression has been documented in multiple human tissues, with notable detection in brain and renal tissues through immunohistochemical analysis . Importantly, VIMP has been detected in both normal brain tissue and renal cancer tissue, suggesting potential differential expression patterns between normal and pathological states. When investigating VIMP expression, researchers should consider:

• ER-rich tissues, given VIMP's established role in ER function

• Tissues under conditions of ER stress, where VIMP function may be modulated

• Comparative analysis between normal and disease-state tissues

• Correlation with other markers of ER function or stress response

Tissue-specific expression patterns may provide insights into VIMP's functional roles in different cellular contexts and disease states.

How should VIMP antibodies be stored to maintain optimal activity?

VIMP antibodies, like the rabbit polyclonal formulations commonly available, are typically provided in a stabilized formulation containing PBS (pH 7.3), sodium azide (0.05%), and glycerol (50%) . For optimal preservation of activity:

• Store antibodies at -20°C as recommended by manufacturers

• Avoid repeated freeze-thaw cycles by aliquoting upon receipt

• When working with the antibody, keep on ice and return to storage promptly

• Monitor for signs of degradation (loss of activity, increased background)

• Check expiration dates and validate antibody performance periodically

Proper storage is critical for maintaining antibody function over time, particularly for applications requiring high sensitivity such as immunohistochemistry of low-abundance targets.

How can I optimize co-immunoprecipitation protocols for studying VIMP protein interactions?

Co-immunoprecipitation (co-IP) is a valuable technique for investigating VIMP protein interactions, such as its documented association with CLIMP-63 . For optimized VIMP co-IP protocols:

• Lysis Buffer Selection: Use mild, non-denaturing buffers (e.g., HEPES-based buffers with 150mM NaCl, 1% NP-40 or 0.5% Triton X-100) to preserve protein-protein interactions while effectively solubilizing membrane proteins like VIMP.

• Pre-clearing Step: Implement a pre-clearing step with protein A/G beads and control IgG to reduce non-specific binding.

• Antibody Binding: Incubate cell lysates with anti-VIMP antibody overnight at 4°C with gentle rotation to maximize specific antigen capture.

• Wash Conditions: Use at least 4-5 washes with decreasing detergent concentrations to reduce background while preserving specific interactions.

• Controls: Always include appropriate controls:

  • Input sample (pre-immunoprecipitation lysate)

  • IgG control immunoprecipitation (non-specific antibody of same isotype)

  • Negative control samples (cells with VIMP knockdown)

Successful co-IP experiments have demonstrated that CLIMP-63 coprecipitates with anti-VIMP antibody but not with control IgG, while α-tubulin does not coprecipitate with VIMP, confirming the specificity of the VIMP-CLIMP-63 interaction .

What approaches can resolve contradictory findings when using different VIMP antibodies?

When faced with contradictory results from different VIMP antibodies, a systematic investigative approach is necessary:

• Epitope Mapping: Determine the specific epitopes recognized by each antibody. Different antibodies may target distinct regions of VIMP, potentially affecting accessibility in certain conformational states or protein complexes.

• Cross-Validation: Employ multiple detection methods (Western blot, immunofluorescence, flow cytometry) to compare antibody performance across platforms.

• Knockout/Knockdown Validation: Test antibodies in VIMP-deficient systems to confirm specificity and rule out cross-reactivity with similar proteins.

• Phosphorylation/Post-translational Modification Sensitivity: Assess whether discrepancies relate to differential detection of modified VIMP forms by performing dephosphorylation experiments or using modification-specific antibodies.

• Protocol Standardization: Standardize all experimental conditions (fixation methods, antigen retrieval, blocking reagents) when comparing antibodies.

• Sequential Epitope Exposure: For challenging samples, try sequential or combinatorial approaches where multiple antibodies are used in series to maximize detection.

Resolution of contradictory findings often reveals important biological insights about target protein states, interactions, or modifications that were previously unrecognized.

How can VIMP antibodies be integrated into multiplexed imaging approaches?

Incorporating VIMP antibodies into multiplexed imaging requires careful consideration of antibody properties and protocol optimization:

• Antibody Selection for Multiplexing:

  • Choose primary antibodies from different host species to prevent cross-reactivity

  • Ensure compatible fixation requirements across all antibodies in the panel

  • Validate each antibody individually before combining

• Sequential Staining Protocols:

  • For challenging combinations, implement sequential staining with careful stripping or quenching between rounds

  • Validate that stripping procedures do not affect tissue morphology or antigen availability

• Spectral Unmixing Considerations:

  • Select fluorophores with minimal spectral overlap

  • Include single-stain controls for accurate spectral unmixing

  • Use automated analysis algorithms to separate closely overlapping signals

• VIMP-Specific Optimization:

  • As VIMP is an ER membrane protein, combine with other organelle markers (e.g., calnexin, PDI for ER, α-tubulin for microtubules) to investigate spatial relationships

  • Consider using super-resolution microscopy techniques for detailed subcellular localization studies

Multiplexed imaging with VIMP antibodies enables comprehensive analysis of its spatial relationships with interacting partners like CLIMP-63 and cellular structures like microtubules in the context of ER organization.

What are common causes of high background when using VIMP antibodies in IHC?

High background in VIMP immunohistochemistry can stem from multiple sources that require systematic troubleshooting:

Successful IHC staining with VIMP antibodies has been demonstrated in paraffin-embedded human brain and renal cancer tissues, indicating these challenges can be overcome with proper protocol optimization .

How can I improve detection sensitivity when working with low VIMP expression samples?

For samples with low VIMP expression, sensitivity enhancement requires a multi-faceted approach:

• Signal Amplification Systems:

  • Implement tyramide signal amplification (TSA) which can increase sensitivity 10-50 fold

  • Consider polymer-based detection systems that carry multiple enzyme molecules per antibody binding event

  • Evaluate quantum dot conjugates for enhanced signal stability and brightness

• Sample Preparation Optimization:

  • Fine-tune fixation protocols to preserve antigenicity while maintaining morphology

  • Test multiple antigen retrieval methods (heat-induced vs. enzymatic; different pH buffers)

  • Minimize time between tissue collection and fixation to prevent protein degradation

• Antibody Enhancement Strategies:

  • Use cocktails of VIMP antibodies targeting different epitopes

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

  • Implement biotin-streptavidin amplification systems

• Instrument Optimization:

  • Use high-sensitivity cameras or photomultiplier tubes

  • Optimize exposure settings and dynamic range

  • Consider confocal microscopy to eliminate out-of-focus fluorescence

• Digital Enhancement:

  • Apply appropriate image processing algorithms (deconvolution, background subtraction)

  • Use quantitative analysis software to detect subtle differences in expression levels

These approaches, used individually or in combination, can significantly improve detection of low-abundance VIMP in challenging samples.

What controls are essential when validating a new lot of VIMP antibody?

Rigorous validation of new VIMP antibody lots requires a comprehensive control strategy:

• Positive Tissue Controls:

  • Include previously validated human brain and renal cancer tissue samples known to express VIMP

  • Process control tissues alongside experimental samples

• Negative Controls:

  • Isotype control antibody at the same concentration as the VIMP antibody

  • Secondary antibody-only controls to assess non-specific binding

  • VIMP-knockout or knockdown samples (when available)

• Peptide Competition:

  • Pre-incubate antibody with excess immunizing peptide to confirm specificity

  • Include both competed and non-competed antibody conditions

• Cross-Batch Comparison:

  • Side-by-side comparison with previously validated lot

  • Quantitative assessment of staining intensity and pattern

• Protocol Verification:

  • Confirm performance across range of dilutions (1:10 to 1:50)

  • Verify compatibility with your specific sample preparation methods

• Functional Validation:

  • Verify expected protein-protein interactions (e.g., VIMP-CLIMP-63)

  • Confirm known biological effects (e.g., ER-MT bundling upon VIMP overexpression)

How can structure-based antibody engineering enhance VIMP antibody performance?

Structure-based antibody engineering offers promising avenues for improving VIMP antibody characteristics through rational design approaches:

• Affinity Maturation: Targeted modifications in complementarity-determining regions (CDRs) can enhance binding affinity to VIMP. This requires structural knowledge of the antibody-antigen complex, which can guide the identification of key residues for modification . Higher-affinity VIMP antibodies would improve detection of low-abundance protein and enhance immunoprecipitation efficiency.

• Specificity Enhancement: Rational engineering can minimize cross-reactivity with related proteins by modifying residues at the antibody-antigen interface. Analysis of antibody binding to VIMP versus potential cross-reactive targets can inform targeted amino acid substitutions that enhance selectivity .

• Stability Improvement: Modifications to framework regions can enhance antibody stability without affecting binding properties. Techniques such as improving hydrophobic core packing, introducing disulfide bonds, or removing aggregation-prone regions can yield VIMP antibodies with extended shelf-life and activity .

• Humanization: For potential therapeutic applications, mouse-derived anti-VIMP antibodies can be humanized through careful grafting of CDRs onto human frameworks. Multiple humanization strategies exist, including selection of human templates based on sequence identity, canonical structure similarity, and stability characteristics .

• Biophysical Property Optimization: Engineering can address challenges such as poor solubility, aggregation tendency, or non-specific binding by modifying surface-exposed residues without compromising antigen recognition.

These engineering approaches require structural data on the antibody-antigen complex, which may be obtained through X-ray crystallography, cryo-EM, or computational modeling techniques.

What emerging technologies could enhance VIMP research beyond traditional antibody applications?

Several cutting-edge technologies are poised to transform VIMP research beyond conventional antibody applications:

• Proximity Labeling Technologies:

  • APEX2 or BioID fusion to VIMP for mapping protein interaction networks

  • Allows identification of transient or weak interactions within the native cellular environment

  • Can reveal novel VIMP functions by identifying previously unknown binding partners

• CRISPR-Based Genomic Tagging:

  • Endogenous tagging of VIMP for live-cell imaging without overexpression artifacts

  • Enables tracking of VIMP dynamics at physiological expression levels

  • Can be combined with engineered antibody fragments for super-resolution microscopy

• Single-Cell Analysis:

  • Single-cell proteomics to examine VIMP expression heterogeneity

  • Spatial transcriptomics to correlate VIMP expression with tissue microenvironment

  • Reveals cell type-specific functions and regulatory mechanisms

• Antibody-Like Alternative Scaffolds:

  • Nanobodies, DARPins, or aptamers targeting VIMP with potentially superior properties

  • Smaller size enables access to epitopes restricted to conventional antibodies

  • Can be genetically encoded for intracellular expression and real-time monitoring

• Computational Approaches:

  • AI-driven epitope prediction for designing optimized VIMP binders

  • Molecular dynamics simulations to understand VIMP conformational changes upon binding

  • Systems biology modeling of VIMP's role in cellular pathways

These emerging approaches complement traditional antibody-based techniques and offer new avenues for understanding VIMP's complex roles in cellular physiology and pathology.

How might VIMP antibodies be employed in studying disease mechanisms?

VIMP antibodies can serve as powerful tools for investigating disease-related mechanisms through several strategic applications:

• Comparative Pathology Studies:

  • Examining VIMP expression and localization across normal versus diseased tissues

  • Validated IHC protocols using VIMP antibodies have already demonstrated applicability in brain tissues and renal cancer samples

  • Correlation of VIMP expression patterns with disease progression markers

• ER Stress Response Investigation:

  • VIMP's role in ER-associated degradation makes it relevant to diseases involving ER stress

  • Antibody-based tracking of VIMP re-localization or expression changes during stress conditions

  • Co-labeling with other ER stress markers to establish temporal relationships

• Cytoskeletal Dynamics in Disease:

  • Given VIMP's interaction with CLIMP-63 and role in ER-microtubule bundling , VIMP antibodies can illuminate alterations in ER-cytoskeleton interactions in neurological disorders

  • Analysis of these dynamics in models of neurodegeneration, where ER-cytoskeleton interactions are frequently disrupted

• Therapeutic Target Validation:

  • Antibody-based confirmation of VIMP accessibility in therapeutic approaches

  • Development of blocking antibodies to interrogate functional consequences of VIMP inhibition

  • Structure-guided design of therapeutic candidates based on epitope mapping with existing antibodies

• Biomarker Development:

  • Quantitative assessment of VIMP levels in patient samples using validated antibodies

  • Correlation of VIMP expression or post-translational modifications with disease outcomes

  • Potential for developing diagnostic or prognostic assays based on VIMP detection

The strategic application of VIMP antibodies across these research areas holds promise for uncovering new disease mechanisms and potential therapeutic approaches.