VIM Recombinant Monoclonal Antibody

What is the molecular target of VIM recombinant monoclonal antibodies?

VIM recombinant monoclonal antibodies target vimentin, a 57-58 kDa intermediate filament protein (IFP) expressed in cells of mesenchymal origin . These antibodies are specifically designed to recognize epitopes on human vimentin (UniProt ID: P08670) without cross-reactivity to other closely related intermediate filament proteins such as desmin, keratin, neurofilament, and glial fibrillary acid protein . Vimentin is highly conserved across species, which explains the broad species reactivity of many VIM antibodies, though notably some clones like VIM/1937R do not react with mouse vimentin .

What are the key differences between various VIM antibody clones?

Different VIM antibody clones vary in their species reactivity, applications, and epitope recognition. For example:

How does vimentin function in cellular physiology?

Vimentin serves multiple critical functions in cells. It is involved in the intracellular transport of proteins between the nucleus and plasma membrane . Unlike other intermediate filament proteins, vimentin is expressed along with desmin during early developmental stages and is later exchanged for tissue-specific IFPs during cellular maturation . Vimentin also plays important roles in steroid synthesis by forming a storage network for steroidogenic cholesterol-containing lipid droplets . When phosphorylated by protein kinases, vimentin filaments break down, activating ATP and myosin light chain-dependent contractile events that facilitate interaction between lipid droplets and mitochondria . Additionally, vimentin can be expressed at the cell surface, mediating cell-cell interactions, tissue repair processes, immune responses, and pathogen recognition .

What are the optimal conditions for using VIM antibodies in immunohistochemistry of formalin-fixed tissues?

For optimal immunohistochemical staining of formalin-fixed tissues with VIM antibodies, heat-induced epitope retrieval is critical. The recommended protocol involves:

• Sectioning formalin-fixed, paraffin-embedded tissues at 4-6 μm thickness

• Performing antigen retrieval by heating tissue sections in 10mM Tris with 1mM EDTA, pH 9.0, for 45 minutes at 95°C

• Cooling sections at room temperature for 20 minutes before antibody application

• Incubating with the primary antibody (e.g., clone VIM, 6430R) for 30 minutes at room temperature

• Following with appropriate detection systems compatible with rabbit monoclonal antibodies

The specific dilution should be experimentally determined for each application, though starting dilutions of 1:100 to 1:500 are typically recommended .

How can researchers optimize VIM antibody performance in flow cytometry?

For intracellular flow cytometry analysis of vimentin:

• Cell fixation and permeabilization are essential steps, as vimentin is primarily a cytoplasmic protein

• Use a fixation/permeabilization kit compatible with intracellular protein detection

• For recombinant rabbit monoclonal antibodies like VIM/1937R, initial dilutions of 1-10 μg/mL are recommended, with further optimization based on cell type and expression level

• Include proper controls:

  • Isotype control (e.g., rabbit IgG) to assess nonspecific binding

  • Positive control cell lines known to express vimentin (e.g., human fibroblast cell lines like LEP-19)

  • Negative control cell lines or tissues (e.g., mouse cells for antibodies that don't cross-react with mouse vimentin)

What considerations are important for western blot applications of VIM antibodies?

For optimal western blot results with VIM antibodies:

• Sample preparation:

  • Use RIPA buffer or other suitable lysis buffers containing protease inhibitors

  • Typical protein loading of 10-30 μg per lane is sufficient for detecting vimentin in most mesenchymal cells

• Electrophoresis and transfer:

  • Vimentin's molecular weight is 57-58 kDa, so standard SDS-PAGE conditions are appropriate

  • Standard transfer conditions to PVDF or nitrocellulose membranes are effective

• Antibody incubation:

  • Starting dilution of 1-2 μg/mL is recommended for primary antibody incubation

  • Overnight incubation at 4°C often yields optimal results with minimal background

• Detection:

  • Both chemiluminescence and fluorescence-based detection systems are compatible

  • The choice of secondary antibody should match the host species (rabbit for most recombinant clones)

How can VIM antibodies be utilized to study epithelial-to-mesenchymal transition (EMT) in cancer research?

Vimentin is a key marker for epithelial-to-mesenchymal transition, making VIM antibodies valuable tools in cancer research . Researchers can:

• Use multiparameter analysis combining VIM antibodies with epithelial markers (E-cadherin, cytokeratins) to identify cells undergoing EMT

• Employ fluorescently conjugated VIM antibodies (e.g., mFluor Violet 610 SE conjugated VIM/1937R) for multicolor flow cytometry or immunofluorescence to quantify EMT in heterogeneous tumor samples

• Correlate vimentin expression with metastatic potential by comparing primary tumors versus metastatic lesions

• Evaluate the efficacy of EMT-targeting compounds by monitoring changes in vimentin expression

Studies have shown that high vimentin expression correlates with increased metastasis in oral squamous cell carcinomas and other tumor types, making quantitative assessment of vimentin critical in translational cancer research .

What are the considerations for using VIM antibodies in distinguishing between tumor types?

While vimentin antibodies alone have limited diagnostic value, they become powerful tools when used in panels with other markers:

• Differential diagnosis approaches:

  • Melanoma vs. carcinoma: Vimentin+/Keratin- pattern suggests melanoma

  • Sarcoma vs. carcinoma: Vimentin+/Keratin- pattern in most sarcomas

  • Lymphoma vs. carcinoma: Vimentin+/CD45+/Keratin- pattern in lymphomas

• Notable exceptions where vimentin negativity is diagnostically valuable:

  • Hepatomas and seminomas typically do not express vimentin

  • This negative reactivity pattern can be more diagnostically useful than positive reactivity in certain contexts

• Antibody selection considerations:

  • Choose clones with appropriate species reactivity for your model system

  • Consider fluorescent conjugates for multiplex analysis with other tumor markers

  • Validate staining patterns in known positive and negative control tissues

How can phosphorylation-specific vimentin antibodies enhance understanding of cytoskeletal dynamics?

Advanced research into vimentin function often requires analysis of its phosphorylation state:

• Vimentin phosphorylation by protein kinases triggers the breakdown of intermediate filaments and activates contractile events dependent on ATP and myosin light chains

• This leads to cytoskeletal reorganization facilitating lipid droplet-mitochondria interactions and cholesterol transport to organelles

• Researchers can use phosphorylation-specific vimentin antibodies alongside total vimentin antibodies to:

  • Track dynamic changes in vimentin organization during cell migration

  • Study the relationship between vimentin phosphorylation and steroid synthesis

  • Investigate how extracellular signals regulate vimentin filament assembly/disassembly

• Methodological considerations include using phosphatase inhibitors during sample preparation and validating phospho-specificity through phosphatase treatment controls

What are common causes of false negative results with VIM antibodies?

Several factors can contribute to false negative results when using VIM antibodies:

• Inadequate antigen retrieval:

  • Insufficient heat treatment (temperature below 95°C)

  • Incorrect buffer composition (pH 9.0 EDTA-based buffers are often optimal)

  • Insufficient retrieval time (less than the recommended 45 minutes)

• Species incompatibility:

  • Using antibodies on incompatible species (e.g., using mouse-negative clones like VIM/1937R on mouse samples)

• Sample preparation issues:

  • Overfixation with formalin can mask epitopes

  • Protein degradation due to delayed fixation

  • Improper permeabilization for intracellular staining in flow cytometry or immunofluorescence

• Technical considerations:

  • Using suboptimal antibody dilutions

  • Insufficient incubation time

  • Using expired or improperly stored antibodies

How should researchers interpret variable vimentin expression patterns in heterogeneous samples?

Interpreting variable vimentin expression requires understanding its biological context:

• In tumor samples:

  • Heterogeneous vimentin expression may indicate varying degrees of EMT or the presence of stromal components

  • Co-staining with epithelial markers helps distinguish tumor cells undergoing EMT from stromal fibroblasts

  • Quantitative assessment of vimentin-positive cell populations across tumor regions can reveal invasion fronts

• In developmental studies:

  • Dynamic expression patterns reflect developmental transitions

  • Vimentin expression precedes tissue-specific IFP expression

  • Co-expression with other IFPs may indicate transitional states

• Statistical approaches:

  • Consider using digital image analysis for quantification of staining intensity and distribution

  • Report both percentage of positive cells and staining intensity

  • For flow cytometry data, analyze median fluorescence intensity rather than just percent positive

What experimental controls are essential for validating VIM antibody specificity?

Proper validation of VIM antibody specificity requires multiple controls:

• Positive tissue controls:

  • Mesenchymal tissues (fibroblasts, endothelial cells)

  • Melanomas and Schwannomas (known to strongly express vimentin)

• Negative tissue controls:

  • Hepatomas and seminomas (typically vimentin-negative)

  • Mouse tissues when using mouse-negative antibody clones

• Cellular controls:

  • Knockdown/knockout validation using siRNA or CRISPR against vimentin

  • Competing peptide assays to confirm epitope specificity

  • Western blot correlation to confirm the 57-58 kDa band corresponds to vimentin

• Technical controls:

  • Isotype controls matched to the antibody's host species and isotype

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

  • Multiple antibody clones targeting different epitopes for confirmation

How are VIM antibodies being used to study vimentin's role in viral infections?

Recent research has revealed vimentin's multifaceted role in viral infections:

• Vimentin is often expressed at the cell surface, playing important roles in:

  • Cell-cell interactions

  • Tissue damage and repair processes

  • Immune response regulation

  • Pathogen recognition

• Activated macrophages secrete phosphorylated vimentin, which can be:

  • Stimulated by pathophysiological factors including oxidized low-density lipoproteins and TNF-alpha

  • Inhibited by anti-inflammatory cytokines like IL-10

• Research applications of VIM antibodies in virology include:

  • Tracking vimentin redistribution during viral entry and replication

  • Investigating vimentin as a potential viral receptor or co-receptor

  • Studying how viruses manipulate the vimentin cytoskeleton during infection

  • Examining vimentin's role in viral assembly and egress

What methodologies combine VIM antibodies with other techniques for studying cytoskeletal dynamics?

Advanced cytoskeletal research often integrates VIM antibodies with complementary techniques:

• Live-cell imaging approaches:

  • Combining fixed-cell VIM antibody staining with live-cell vimentin-fluorescent protein fusions

  • Correlative light and electron microscopy to relate vimentin organization to ultrastructural features

  • Super-resolution microscopy for detailed vimentin filament organization

• Biochemical interaction studies:

  • Immunoprecipitation with VIM antibodies followed by mass spectrometry to identify interaction partners

  • Proximity ligation assays to detect vimentin interactions with other cytoskeletal components

  • ChIP-seq approaches to study vimentin's potential roles in gene regulation

• Functional studies:

  • Combining vimentin localization with traction force microscopy

  • Correlating vimentin organization with cell migration tracking

  • Integrating vimentin quantification with measurements of organelle positioning

How might VIM antibodies contribute to therapeutic development targeting intermediate filaments?

Vimentin is emerging as a potential therapeutic target, particularly in cancer:

• Multiple vimentin-targeting compounds are currently in cancer-related clinical trials

• VIM antibodies are essential tools for:

  • Target validation in preclinical models

  • Measuring compound efficacy in reducing vimentin expression or organization

  • Developing companion diagnostics to identify patients likely to respond to vimentin-targeting therapies

  • Monitoring on-target effects in normal tissues expressing vimentin

• Important considerations:

  • Given vimentin's multifunctional role, careful examination of effects on non-malignant cells is necessary

  • VIM antibodies can help distinguish between on-target inhibition and off-target cytotoxicity

  • Multiplex analyses combining vimentin with cell viability, proliferation, and other markers provide comprehensive assessment of therapeutic effects