Vimentin PAT5H8AT Antibody

What is Vimentin PAT5H8AT Antibody and what are its general research applications?

Vimentin PAT5H8AT Antibody (CPAB0668) is a mouse monoclonal antibody (IgG1 isotype) derived from hybridization of mouse F myeloma cells with spleen cells from BALB/c mice immunized with recombinant human Vimentin protein (amino acids 1-466) purified from E. coli . This antibody has been purified from mouse ascitic fluids using protein-A affinity chromatography to ensure high specificity and minimal background interference . The antibody has been validated for several research applications including Western blotting, ELISA (Enzyme-Linked Immunosorbent Assay), and flow cytometry, making it versatile for multiple experimental approaches .

As vimentin plays essential roles in cell migration, adhesion, and signaling pathways, this antibody serves as a valuable tool for studying both normal cellular processes and pathological conditions where vimentin expression is altered . Its high reactivity with human vimentin makes it particularly useful for cancer research, fibrosis studies, and investigations into cellular structure and function .

Why is vimentin an important target in biomedical research?

Vimentin is a 57 kDa type III intermediate filament protein that forms part of the cytoskeleton in mesenchymal cells. Its importance in biomedical research stems from several key characteristics:

• Expression profile: Vimentin is primarily expressed in cells of mesenchymal origin, including fibroblasts, endothelial cells, and certain immune cells. Its expression is dramatically altered during epithelial-mesenchymal transition (EMT), a process critical in development and disease .

• Cancer relevance: Vimentin expression is highly upregulated in metastatic cancers, and its increased levels correlate with poor patient prognoses . Studies show that vimentin is required for the progression of non-small cell lung cancer (NSCLC) and other malignancies .

• Structural roles: Beyond being a structural protein, vimentin actively participates in cell signaling, migration, and adhesion - all processes crucial for understanding disease mechanisms .

• Post-translational modifications: Vimentin undergoes numerous modifications including citrullination, phosphorylation, and others that affect its function in both physiological and pathological conditions .

• Localization dynamics: Vimentin can be found both intracellularly and on the cell surface (EDV - extracellular domain vimentin), with the latter being particularly relevant in cancer cells and potential immunotherapy applications .

Research indicates that vimentin has a "Jekyll/Hyde" aspect - internal cytoplasmic (Jekyll) vimentin acts normally, whereas cell surface (Hyde) vimentin reflects oncological circumstances with a remodeled cytoskeleton .

What are the optimal sample preparation methods for immunofluorescence with PAT5H8AT antibody?

Achieving high-quality immunofluorescence results with PAT5H8AT antibody requires careful attention to sample preparation:

For cultured cells:

• Fixation: Use 4% paraformaldehyde for 15 minutes at room temperature to preserve vimentin structure and epitope accessibility. For certain applications, methanol fixation (-20°C for 10 minutes) may provide better results for cytoskeletal antigens.

• Permeabilization: Treat with 0.1-0.2% Triton X-100 for 5-10 minutes when studying intracellular vimentin. Importantly, when examining cell surface vimentin, avoid permeabilization to ensure only extracellular epitopes are detected .

• Blocking: Incubate with 5% normal serum (goat or donkey) with 0.1% BSA for 30-60 minutes to minimize non-specific binding.

For tissue sections:

• Fresh frozen sections should be fixed with 4% PFA post-sectioning, while FFPE sections require standard deparaffinization.

• Antigen retrieval: Heat-induced epitope retrieval using citrate buffer (pH 6.0) or Tris-EDTA (pH 9.0) is essential for optimal vimentin detection.

• For brain tissue samples, special consideration should be given to autofluorescence reduction using 0.1% Sudan Black B or commercial quenchers, particularly important when studying vimentin in reactive astrocytes .

Antibody incubation parameters:

• Primary antibody (PAT5H8AT): Dilute 1:50-1:200 and incubate overnight at 4°C for best results

• Secondary antibody: Use fluorophore-conjugated anti-mouse IgG at manufacturer's recommended dilution

• When co-staining for citrullinated vimentin forms, sequential staining protocols may yield better results, as demonstrated in studies examining citrullinated vimentin in reactive astrocytes .

How can I optimize Western blotting protocols for detecting vimentin with PAT5H8AT antibody?

Western blotting optimization for vimentin detection requires consideration of several key parameters:

Sample preparation:

• Use RIPA buffer supplemented with protease inhibitors for efficient vimentin extraction. When studying post-translational modifications like citrullination, include specific inhibitors of modifying enzymes (e.g., PAD inhibitors for citrullination studies) .

• Sonication can help break down vimentin filaments for better detection, especially in dense cytoskeletal preparations.

Electrophoresis and transfer conditions:

• 10% SDS-PAGE gels are typically suitable for resolving vimentin (57 kDa)

• For detecting cleaved vimentin fragments, use gradient gels (4-15%) that can resolve lower molecular weight bands, as observed in studies of caspase-mediated vimentin cleavage .

• Semi-dry transfer with methanol-containing buffer often provides optimal vimentin transfer.

Blocking and antibody incubation:

• Block membranes with 5% non-fat milk or BSA in TBST

• Optimize primary antibody dilution (starting at 1:1000, adjusting as needed)

• Incubate with PAT5H8AT antibody overnight at 4°C for best results

Special considerations:

• When examining citrullinated vimentin, Western blotting analysis has shown both full-length and cleaved forms in cerebral cortices of sporadic Creutzfeldt-Jakob disease patients .

• Citrullination promotes resistance to caspase-3 and caspase-9-mediated fragmentation of vimentin, which may affect the banding pattern observed .

• For detecting cell surface vimentin, surface protein isolation techniques prior to Western blotting may be required to distinguish from cytoplasmic vimentin pools .

What protocols are recommended for co-immunoprecipitation with PAT5H8AT antibody?

Effective co-immunoprecipitation (Co-IP) with PAT5H8AT antibody to identify vimentin-interacting proteins requires careful optimization:

Buffer selection and lysis conditions:

• Use immunoprecipitation lysis buffer containing protease inhibitors. Lyse cells for approximately 30 minutes on ice to maintain protein-protein interactions .

• For preserving weak or transient interactions, consider incorporating reversible cross-linking agents.

Immunoprecipitation approach:

• Add agar glycoprotein beads A/G homogenate to the lysate and rinse three times with lysis buffer .

• Reserve 10% of the total volume as input control, then divide the remaining sample .

• Incubate one portion with normal mouse IgG (negative control) and the other with PAT5H8AT antibody. Slow shaking overnight at 4°C ensures optimal antibody-antigen binding .

• After overnight incubation, add pre-washed agarose beads and incubate for an additional 2 hours at 4°C .

• Wash the immunoprecipitates thoroughly to remove non-specific binding.

Controls and validation:

• Input control (10% of total lysate) is essential for comparing protein levels

• IgG isotype control identifies non-specific binding

• Reverse Co-IP with antibodies against suspected interacting partners strengthens confidence in results

Analysis approaches:

• Western blotting with specific antibodies can identify known or suspected interaction partners

• For unbiased discovery, mass spectrometry analysis of immunoprecipitates can reveal novel vimentin-interacting proteins

• Chromatin immunoprecipitation followed by sequencing (ChIP-seq) has been successfully used to identify genes interacting with vimentin in polyploid giant cancer cells, with subsequent GO and KEGG enrichment analyses revealing functional pathways .

How can I use PAT5H8AT antibody to study vimentin's role in cancer progression?

Multiple experimental approaches utilizing PAT5H8AT antibody can advance our understanding of vimentin's role in cancer:

Tissue expression analysis:

• Immunohistochemistry using PAT5H8AT allows evaluation of vimentin expression patterns in tumor versus normal tissue. Research shows vimentin expression is altered in various cancer types, including colorectal carcinomas where aberrant vimentin expression correlates with activated Notch and TGFβ signaling pathways .

• Immunofluorescence co-staining with EMT markers (E-cadherin, N-cadherin) can reveal correlations between vimentin expression and the EMT process crucial for metastasis .

Functional studies:

• Combine antibody staining with migration/invasion assays to correlate vimentin expression levels with metastatic potential. Studies have demonstrated that ablation of vimentin expression inhibits migration and invasion of colon and breast cancer cell lines .

• In vivo models comparing vimentin-expressing versus vimentin-knockout cancers show that vimentin is required for tumor progression. For example, LSL-KrasG12D; Tp53fl/fl mice with vimentin knockout (KPV−/−) demonstrate attenuated tumor growth and improved survival compared to vimentin-expressing (KPV+/+) counterparts .

Cell surface vimentin (EDV) targeting:

• PAT5H8AT can be used in non-permeabilized immunostaining to specifically detect cell surface vimentin, which appears to be a promising immunotherapy target .

• Two lymph node-derived antibodies (pritumumab and RM2) targeting different epitopes of cell surface vimentin have shown therapeutic potential, suggesting oligoclonal targeting of vimentin may enhance immune responses against cancer cells .

Therapeutic intervention assessment:

• Evaluate the effects of vimentin-targeting compounds, such as withaferin A (WFA), which disrupts vimentin intermediate filaments and has been shown to suppress tumor growth and reduce tumor burden in lung cancer models .

• Monitor changes in vimentin expression and organization following treatment with potential anti-cancer therapies.

What methods can detect different vimentin post-translational modifications using PAT5H8AT?

Vimentin undergoes numerous post-translational modifications (PTMs) that regulate its function and structure. Detection methods combining PAT5H8AT with specialized approaches include:

Citrullination detection:

• Citrullination, the calcium-dependent conversion of arginine to citrulline by peptidylarginine deiminase (PAD) enzymes, can be detected using specialized antibodies targeting citrullinated epitopes .

• Studies show vimentin is highly citrullinated at R440 and R450 positions in reactive astrocytes in scrapie-infected mice and in cerebral cortices of sporadic Creutzfeldt-Jakob disease patients .

• Sequential immunoprecipitation with PAT5H8AT followed by anti-citrullinated protein antibodies can identify citrullinated vimentin pools.

Phosphorylation analysis:

• Phospho-specific vimentin antibodies can be used in combination with PAT5H8AT to identify phosphorylated vimentin subpopulations.

• Treatment with phosphatase inhibitors during sample preparation is essential to preserve phosphorylation states.

• Phosphorylation significantly affects vimentin organization and solubility, with implications for cell division and migration.

Biochemical fractionation for localization studies:

• Citrullination leads to increased cytoplasmic and integral membrane/organelle vimentin enrichment, indicating changes in intrinsic solubility and distribution .

• Differential extraction protocols can separate vimentin pools based on their solubility properties, which are affected by post-translational modifications.

Mass spectrometry approaches:

• Immunoprecipitate vimentin using PAT5H8AT, then analyze by mass spectrometry to identify and quantify various modifications.

• This approach allows simultaneous detection of multiple PTMs and their relative abundance in different cellular contexts.

How can I distinguish between cytoplasmic and cell surface vimentin?

Distinguishing between cytoplasmic (intracellular) and cell surface vimentin (EDV) requires specialized techniques:

Cell surface selective labeling:

• Non-permeabilized immunostaining: Fix cells with 2-4% paraformaldehyde without permeabilization, then incubate with PAT5H8AT. Only cell surface vimentin will be accessible to the antibody under these conditions .

• Compare with permeabilized samples (using 0.1% Triton X-100) to visualize total vimentin distribution.

Flow cytometry approach:

• Analyze live, non-permeabilized cells with PAT5H8AT to detect surface vimentin.

• In parallel, analyze fixed and permeabilized cells to measure total vimentin.

• Calculate the ratio of surface to total vimentin to quantify relative surface expression.

Biochemical fractionation:

• Isolate cell surface proteins using biotinylation and streptavidin pull-down.

• Compare with total cell lysate by Western blotting using PAT5H8AT.

• Research indicates that cell surface vimentin (EDV) is particularly relevant in cancer cells that have undergone EMT and may serve as a target for immunotherapy .

Functional significance:

• Research has described a "Jekyll/Hyde" aspect of vimentin - intracellular (Jekyll) vimentin functions normally in cytoskeletal maintenance, while cell surface (Hyde) vimentin reflects oncological circumstances with a remodeled cytoskeleton .

• The recognition of cell surface vimentin suggests an innate auto-antigenic natural human immune response, possibly functioning in immunosurveillance .

• Human lymph nodes appear to process epithelial tumor cells in such a way that surface vimentin (EDV) becomes a recognized target, regardless of the tumor type .

What approaches can study vimentin's role in neurological disorders?

Vimentin plays important roles in the central nervous system, particularly in astrocytes and during neuroinflammation:

Reactive astrocyte studies:

• Immunohistochemistry with PAT5H8AT can detect vimentin in brain tissue samples.

• Co-staining with GFAP and YKL-40 helps identify reactive astrocytes, where citrullinated vimentin is prominently expressed .

• Studies have shown citrullinated vimentin acts as a specific indicator for the reactive state of astrocytes under abnormal neurological conditions such as prion diseases .

Analysis of vimentin modifications in neurological contexts:

• Vimentin citrullination has emerged as a pathophysiological outcome of autoimmune diseases and neurodegeneration .

• Western blotting has revealed both full-length and cleaved forms of citrullinated vimentin in cerebral cortices of sporadic Creutzfeldt-Jakob disease patients .

• Biochemically, citrullination promotes resistance to caspase-3 and caspase-9-mediated fragmentation of vimentin, potentially affecting protein turnover in neural cells .

Methodological approaches:

• Specialized monoclonal antibodies targeting vimentin citrullinated at positions R440 or R450 have been developed for studying prion diseases in animal models and patients .

• Immunofluorescent staining combined with confocal microscopy can reveal subcellular distribution changes in vimentin following citrullination - specifically increased cytoplasmic and integral membrane/organelle vimentin enrichment .

• Brain tumor research can utilize PAT5H8AT to analyze vimentin expression in different tumor types and correlate with invasiveness.

Blood-brain barrier considerations:

• Some anti-vimentin antibodies, such as pritumumab, can cross the blood-brain barrier (BBB). Orthotopic brain tumors in mice were successfully imaged within 4 hours of antibody administration, suggesting rapid BBB passage .

• This property may be related to the antibody's isoelectric point (8.6 for pritumumab), as a high isoelectric point may contribute to efficient BBB penetration .

How does vimentin interact with chromatin and what methods can examine these interactions?

Investigating vimentin-chromatin interactions requires specialized techniques that can be performed using PAT5H8AT antibody:

Chromatin Immunoprecipitation (ChIP) approaches:

• Protocol adaptations for vimentin ChIP include optimized crosslinking conditions (1-2% formaldehyde for 10-15 minutes) and appropriate sonication to generate 200-1000 bp fragments .

• ChIP assays have been successfully performed to identify genes interacting with vimentin in polyploid giant cancer cells .

• Sequencing libraries prepared from ChIP samples allow genome-wide identification of vimentin-associated chromatin regions .

Data analysis and biological significance:

• Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses help identify functional pathways associated with vimentin-bound genes .

• The location of peaks around transcription start sites can predict protein-gene interaction sites, providing insights into vimentin's potential role in transcriptional regulation .

Nuclear localization studies:

• Biochemical fractionation can separate chromatin-bound, nuclear soluble, and cytoplasmic vimentin pools for comparative analysis.

• Immunofluorescence microscopy with nuclear counterstains can visualize vimentin's nuclear localization patterns.

• Research has demonstrated molecular mechanisms of vimentin nuclear localization associated with polyploid giant cancer cells, suggesting regulatory roles beyond cytoskeletal functions .

Co-immunoprecipitation studies:

• Immunoprecipitation with PAT5H8AT followed by analysis for chromatin-associated proteins can identify nuclear interaction partners.

• These approaches have shown that vimentin may synergistically regulate polyploid giant cancer cells to generate daughter cells with enhanced invasive and metastatic capabilities .

What is the potential of vimentin as an immunotherapy target and how can PAT5H8AT antibody contribute to this research?

Vimentin shows promising potential as an immunotherapy target, particularly in its cell surface form:

Natural immune responses to vimentin:

• Studies indicate humans make natural antibodies to vimentin, particularly when it appears on the cell surface .

• Two separate human immune responses to vimentin have been identified in cancer patients - pritumumab and RM2 antibodies - both derived from sentinel lymph nodes .

• This natural immune response to extracellular domain vimentin (EDV) may be oligoclonal, with multiple antibodies generated to different epitopes .

Therapeutic applications:

• PAT5H8AT can be used to validate vimentin expression in potential target tissues and to monitor therapeutic responses.

• Antibody cocktails targeting different vimentin epitopes may be more effective than monotherapy, reflecting the natural oligoclonal oncology response .

• Vimentin-targeting antibodies could be developed into therapeutic agents, as demonstrated by pritumumab, which has shown promise in clinical applications .

Experimental approaches:

• Use PAT5H8AT for screening patient samples to identify those likely to respond to vimentin-targeted therapy.

• Develop in vitro and in vivo models to test vimentin-targeting therapeutic approaches.

• Immunohistological data suggests all forms of solid tumors of epithelial origin are EDV-positive, indicating broad potential applicability .

Blood-brain barrier considerations:

• Some anti-vimentin antibodies like pritumumab can cross the blood-brain barrier, making them potentially valuable for targeting brain tumors .

• Engineered antibodies with appropriate isoelectric points might enhance BBB penetration for neurological applications .

How does vimentin contribute to epithelial-mesenchymal transition and metastasis?

Vimentin plays critical roles in epithelial-mesenchymal transition (EMT), a process essential for cancer metastasis:

Vimentin's functions in EMT:

• Serves as a canonical marker of mesenchymal phenotype, with its expression dramatically increased during EMT .

• Facilitates cytoskeletal reorganization required for the morphological changes characteristic of EMT.

• Promotes cell motility and invasion, with studies showing that ablation of vimentin expression inhibits migration and invasion of colon and breast cancer cell lines .

Structural contributions to metastasis:

• Vimentin and detyrosinated microtubules provide structural support for the extensive microtentacles observed in detached tumor cells, offering a mechanism to promote successful metastatic spread .

• Research in mouse models demonstrates that vimentin knockout attenuates tumor growth and improves survival in lung cancer models .

Signaling pathway interactions:

• Primary colorectal carcinomas displaying aberrant expression of vimentin have activated Notch and TGFβ signaling pathways .

• These pathways further reinforce the mesenchymal phenotype, creating a feed-forward loop that maintains invasive capabilities.

Research approaches using PAT5H8AT:

• Monitor vimentin expression changes during EMT using Western blotting and immunofluorescence.

• Perform time-course analysis following EMT induction (via TGF-β, hypoxia, etc.) to track vimentin reorganization.

• Combine vimentin staining with migration/invasion assays to correlate vimentin levels with metastatic potential.

• Use withaferin A (WFA), which disrupts vimentin intermediate filaments, to assess how vimentin disruption affects tumor growth and metastasis .

What are the future directions for vimentin research using PAT5H8AT antibody?

The versatility of PAT5H8AT antibody positions it as a valuable tool for advancing vimentin research across multiple frontiers:

Post-translational modification mapping:

• Detailed characterization of vimentin modifications in different disease contexts, particularly in cancer and neurological disorders, represents a promising research direction.

• Understanding how citrullination, phosphorylation, and other modifications affect vimentin's function and localization will provide insights into its diverse roles .

Therapeutic target validation:

• Cell surface vimentin (EDV) appears ready for "immunotherapy primetime" according to recent research .

• The identification of natural human immune responses to vimentin in cancer patients suggests clinical relevance that merits further investigation .

• Antibody cocktails targeting different vimentin epitopes may offer enhanced therapeutic potential by mimicking the natural oligoclonal response .

Nuclear functions exploration:

• The emerging understanding of vimentin's chromatin interactions and potential role in gene regulation opens new research avenues .

• ChIP-seq and related approaches can further elucidate how vimentin contributes to gene expression programs in normal and disease states .

Cancer progression mechanisms:

• Further investigation into how vimentin facilitates tumor cell invasion and metastasis will enhance our understanding of cancer biology.

• The observed requirement for vimentin in lung cancer progression suggests it may play similar roles in other cancer types that warrant investigation .

Neurological disorder connections:

• Citrullinated vimentin's role as a specific indicator for reactive astrocytes under abnormal neurological conditions highlights the need for further research in this area .

• The development of specialized antibodies to different modified forms of vimentin will facilitate deeper investigation of its roles in neurodegeneration .