Phospho-VIM (S56) Antibody

What is Phospho-VIM (S56) Antibody and what does it detect?

Phospho-VIM (S56) Antibody specifically recognizes vimentin protein when phosphorylated at serine 56 position . Vimentin is a class-III intermediate filament found in various non-epithelial cells, especially mesenchymal cells, and plays critical roles in cell structure and function . This antibody is typically produced by immunizing animals with a synthetic phosphopeptide corresponding to residues surrounding Ser56 of human vimentin protein .

Vimentin is attached to the nucleus, endoplasmic reticulum, and mitochondria, either laterally or terminally . It plays important roles in:

• Cell directional movement and orientation

• Cell sheet organization

• Golgi complex polarization at the cell migration front

• Protecting SCRIB from proteasomal degradation

• Stabilization of type I collagen mRNAs (CO1A1 and CO1A2) in conjunction with LARP6

The antibody enables researchers to specifically study this post-translational modification that regulates vimentin's functions in normal and pathological conditions.

What applications are suitable for Phospho-VIM (S56) Antibody research?

Phospho-VIM (S56) Antibody has been validated for multiple research applications:

The versatility of these applications allows researchers to examine phospho-vimentin expression from multiple analytical perspectives. When using this antibody for the first time in a particular application, validation using appropriate positive and negative controls is strongly recommended .

What is the relationship between vimentin S56 phosphorylation and cell cycle regulation?

Vimentin phosphorylation at S56 plays a crucial role in cell cycle progression and regulation:

During mitosis, CDK1 specifically phosphorylates vimentin at Ser56 . This phosphorylation creates a binding site for Polo-like kinase (PLK) . After binding, PLK further phosphorylates vimentin at Ser83, which is believed to function as a "memory phosphorylation site" that plays a regulatory role in vimentin filament disassembly during cell division .

Phosphorylation of vimentin is significantly enhanced during cell division, coinciding with dramatic reorganization of vimentin filaments . This dynamic remodeling is essential for proper segregation of cellular components during mitosis.

Methods to study cell cycle-dependent phosphorylation include:

• Synchronized cell cultures with timed sampling

• Flow cytometry co-staining for DNA content and phospho-vimentin

• Immunofluorescence microscopy with cell cycle phase markers

• Live cell imaging with fluorescently-tagged vimentin constructs

Research has shown that disrupting the normal phosphorylation status of vimentin-S56 (either through stabilizing or preventing phosphorylation) leads to multinucleation, suggesting its crucial role in maintaining genomic stability during cell division .

How does vimentin S56 phosphorylation impact cancer stem cell properties?

Recent research has uncovered significant connections between vimentin S56 phosphorylation and cancer stem cell (CSC) properties:

Studies using phosphomimetic mutants (VIM-S56E) have demonstrated that stabilizing vimentin S56 phosphorylation leads to multinucleation and inhibits stem-like cell properties . Specifically, FiVe1 treatment or expression of phosphomimetic VIM-S56E inhibited stemness as characterized by mammosphere formation assays .

Interestingly, when CSCs were enriched through various methods such as:

• Growth in serum-free suspension culture

• CD44hi/CD24low sorting by flow cytometry

The CSC-enriched populations showed significantly higher levels of multinucleation after stabilization of vimentin S56 phosphorylation compared to non-enriched cultures . For example, in one study, 4T1 TET-on VIM-S56E cells grown in suspension culture had 2.5-fold more multinucleated cells than non-enriched cells grown in monolayer culture (p = 0.0020) .

Similarly, CSC-enriched CD44hi/CD24low sorted cells showed higher levels of multinucleation after FiVe1 treatment compared to non-CSC CD44low/CD24hi populations:

• 3.6-fold increase for HMLER cells (p = 0.0003)

• 3.5-fold increase for HCC1806 cells (p = 0.0366)

These findings suggest that vimentin S56 phosphorylation is a potential therapeutic target for eliminating cancer stem cells, which are often responsible for tumor recurrence and therapy resistance.

What are the recommended experimental controls when using Phospho-VIM (S56) Antibody?

Proper experimental controls are essential for generating reliable data with Phospho-VIM (S56) Antibody:

Positive Controls:

• Cell lines with known phospho-vimentin S56 expression, such as:

  • MCF-7, NIH-3T3, or H9C2 cell lines treated with UV for 24h

  • HeLa cells for flow cytometry applications

• Samples from cells treated with agents that induce phosphorylation:

  • UV treatment

  • Doxorubicin (1 μM for 24h has been validated)

Negative Controls:

• Phosphatase-treated samples to demonstrate signal specificity

• Cells expressing phospho-ablative mutant (S56A) of vimentin

• Primary antibody omission, replacing with buffer or isotype control

• For IHC, using PBS instead of primary antibody as demonstrated in validated protocols

Specificity Controls:

• Peptide competition assays with phosphorylated and non-phosphorylated peptides

• Cross-validation with multiple antibody clones targeting the same site

• Western blotting to confirm antibody specificity by molecular weight (expected at approximately 57 kDa)

• Testing reactivity against non-phosphorylated vimentin to confirm phospho-specificity

Implementing these controls helps ensure that observed signals genuinely represent phosphorylated vimentin at S56 rather than non-specific binding or artifacts.

How does vimentin S56 phosphorylation differ across normal and cancer tissues?

Phosphorylation patterns of vimentin at S56 show notable differences between normal and cancerous tissues:

In normal tissues, vimentin S56 phosphorylation is tightly regulated and primarily observed during specific cellular processes such as mitosis and neutrophil secretion . Phosphorylation by CDK5 at S56 occurs during neutrophil secretion in the cytoplasm .

In cancer tissues, several important differences have been observed:

• Colorectal cancer specimens have shown altered phospho-vimentin expression, with positive cases defined as those with more than 5% phospho-vimentin-stained cells

• Hybrid epithelial/mesenchymal (E/M) cancer cells exhibit high levels of vimentin-S56 phosphorylation compared to purely epithelial or mesenchymal cells

• Cancer stem cell populations show particularly high sensitivity to disruption of vimentin S56 phosphorylation status

Methods for detecting these differences include:

• Immunohistochemistry on tissue microarrays or tissue sections

• Western blotting of tissue lysates

• Cell-based ELISA for high-throughput quantification

• Multiplexed immunofluorescence to correlate with other markers

The observed differences in phosphorylation patterns may contribute to cancer progression, metastasis, and therapy resistance, making phospho-vimentin a potential biomarker and therapeutic target.

What optimization steps are required for Western blotting with Phospho-VIM (S56) Antibody?

Optimizing Western blotting protocols for Phospho-VIM (S56) Antibody requires attention to several critical parameters:

Sample Preparation:

• Fresh sample collection with immediate addition of phosphatase inhibitors is crucial

• Standardize protein extraction methods, preferably using buffers containing:

  • Phosphatase inhibitors (sodium fluoride, sodium orthovanadate)

  • Protease inhibitors

  • Detergents appropriate for cytoskeletal proteins

Gel Electrophoresis:

• Use SDS-PAGE gels with appropriate concentration (typically 10-12%) for optimal resolution of vimentin (57 kDa)

• Include molecular weight markers spanning 40-70 kDa range

• Load equal amounts of protein (typically 20-50 μg per lane)

Transfer and Blocking:

• Optimize transfer conditions for intermediate filament proteins

• PVDF membranes are often preferred over nitrocellulose for phospho-epitopes

• Block with 5% BSA in TBST rather than milk (milk contains phospho-proteins that may increase background)

Antibody Incubation:

• Starting dilution of 1:1000 is recommended for most Western blotting applications

• Optimize primary antibody incubation time and temperature (typically overnight at 4°C)

• Use TBS-T with 5% BSA for antibody dilution

Detection:

• Use HRP-conjugated secondary antibodies at appropriate dilution (typically 1:2000-1:5000)

• For enhanced sensitivity, consider chemiluminescent substrate systems

• When possible, use quantitative detection methods such as fluorescent secondary antibodies

Validated Western blot results have shown detection of phospho-vimentin (S56) in:

• MCF-7 cells treated with UV (24h)

• NIH-3T3 cells treated with UV (24h)

• H9C2 cells treated with UV (24h)

What methods can be used to functionally study the impact of vimentin S56 phosphorylation?

Several sophisticated methods can help researchers understand the functional significance of vimentin S56 phosphorylation:

Genetic Approaches:

• Expression of phosphomimetic mutants (S56E) to simulate constitutive phosphorylation

• Expression of phospho-ablative mutants (S56A) to prevent phosphorylation

• CRISPR/Cas9 gene editing to modify endogenous vimentin phosphorylation sites

• Doxycycline-inducible expression systems for temporal control

Pharmacological Approaches:

• Treatment with FiVe1, which affects vimentin phosphorylation status

• Kinase inhibitors to block phosphorylation pathways (CDK5, CDK1)

• Phosphatase inhibitors to maintain phosphorylation status

Functional Assays:

• Mammosphere formation assays to assess stemness properties

• Cell migration and invasion assays to assess metastatic potential

• Mitotic index and multinucleation quantification to assess cell division defects

• Cell cycle analysis by flow cytometry to determine cell cycle distribution

Advanced Imaging:

• Live-cell imaging with fluorescently tagged vimentin to track dynamic changes

• Super-resolution microscopy to visualize filament organization

• FRET-based biosensors to detect phosphorylation events in real-time

Research has shown that disrupting vimentin S56 phosphorylation through either phosphomimetic (S56E) or phospho-ablative (S56A) mutations leads to multinucleation in hybrid E/M cancer cells, with an average 6.7-fold increase for S56E and 3.9-fold increase for S56A . These approaches help elucidate the biological significance of this post-translational modification in various cellular contexts.

How does vimentin S56 phosphorylation interact with other post-translational modifications?

Vimentin undergoes multiple post-translational modifications that interact in complex ways:

Phosphorylation at Multiple Sites:

• Besides S56, vimentin can be phosphorylated at S39, S72, and S83, each with distinct functions

• During mitosis, CDK1 phosphorylates vimentin at S56, creating a binding site for PLK, which then phosphorylates vimentin at S83

• This sequential multi-site phosphorylation creates a regulatory cascade

• Phosphorylation by different kinases (CDK5, STK33, PKN1) occurs in different cellular contexts

Interplay with Other Modifications:

• O-glycosylation: Occurs during cytokinesis at sites identical or close to phosphorylation sites, potentially interfering with phosphorylation status

• S-nitrosylation: Induced by interferon-gamma and oxidatively-modified low-density lipoprotein, possibly involving the iNOS-S100A8/9 transnitrosylase complex

• Tyrosine phosphorylation: Vimentin can be phosphorylated on tyrosine residues by SRMS

Regulatory Mechanisms:

• Phosphorylation by PKN1 inhibits the formation of vimentin filaments

• Filament disassembly during mitosis is promoted by phosphorylation at S56 as well as by nestin

• Competition between phosphorylation and O-glycosylation at similar sites may serve as a regulatory switch

Understanding these complex interactions requires sophisticated analytical approaches including:

• Mass spectrometry to identify multiple modifications simultaneously

• Antibodies specific to different phosphorylation sites

• Mutational analysis to determine the hierarchy of modifications

• Temporal analysis to map modification sequences

These interactions create a "modification code" that fine-tunes vimentin function in different cellular contexts.

What are the methodological considerations for immunofluorescence using Phospho-VIM (S56) Antibody?

Successful immunofluorescence with Phospho-VIM (S56) Antibody requires careful attention to several methodological aspects:

Fixation and Permeabilization:

• Paraformaldehyde fixation (typically 4%) is commonly used but may require optimization

• Methanol fixation can sometimes better preserve phospho-epitopes

• Permeabilization with 0.1-0.5% Triton X-100 or 0.1% saponin is typically suitable

• Avoid overfixation, which can mask epitopes

Blocking and Antibody Incubation:

• Use appropriate blocking buffer (typically 1-5% BSA or serum)

• Recommended primary antibody dilution is typically 1:200

• Incubate at 4°C overnight for optimal signal-to-noise ratio

• Include phosphatase inhibitors in buffers to preserve phosphorylation

Signal Detection:

• Use fluorescently-labeled secondary antibodies appropriate for the host species (rabbit for most Phospho-VIM (S56) antibodies)

• Consider signal amplification for low-abundance phospho-epitopes

• Include DAPI or similar nuclear counterstain

• Mount in anti-fade medium to prevent photobleaching

Controls and Validation:

• Include cells known to have high phospho-vimentin S56 levels as positive controls

• Include phosphatase-treated samples as negative controls

• Consider co-staining with total vimentin antibody to normalize signal

• Use z-stack imaging to properly capture filamentous structures

Analysis Considerations:

• Quantify signal intensity using appropriate software

• Analyze subcellular distribution patterns

• Consider co-localization with other markers

• Compare patterns between different cell types or treatments

Immunofluorescence has successfully shown cytoplasmic and nuclear staining patterns for phospho-vimentin (S56) in various cell types, providing valuable information about its subcellular distribution and regulation .

How can Phospho-VIM (S56) Antibody be integrated into multiplexed immunofluorescence studies?

Integrating Phospho-VIM (S56) Antibody into multiplexed immunofluorescence requires strategic planning:

Panel Design:

• Choose complementary markers based on research questions:

  • Total vimentin to normalize phospho-signal

  • Cell cycle markers (cyclin B1, pH3) to correlate with mitotic status

  • EMT markers (E-cadherin, N-cadherin) to study transition states

  • Stemness markers (CD44, SOX2) for cancer stem cell studies

Technical Considerations:

• Ensure primary antibodies are raised in different host species

• If using multiple rabbit antibodies, consider:

  • Sequential staining with complete elution between rounds

  • Directly conjugated primary antibodies

  • Tyramide signal amplification with heat-mediated antibody removal

• Optimize antibody concentration for each marker individually before multiplexing

Controls for Multiplexed Staining:

• Single-color controls to establish spectral properties

• Fluorescence-minus-one (FMO) controls to set thresholds

• Isotype controls to assess non-specific binding

• Peptide competition controls for phospho-specific antibodies

Sample Processing:

• Antigen retrieval methods may be needed, especially for FFPE samples (Cell Conditioning 1 buffer has been used successfully)

• Consider automated staining platforms for reproducibility

• Test fixation protocols that preserve all antigens of interest

Imaging and Analysis:

• Use spectral imaging systems to separate overlapping fluorophores

• Consider multispectral imaging platforms (e.g., Vectra, Mantra)

• Implement quantitative image analysis using software like HALO, QuPath, or CellProfiler

• Analyze co-localization and spatial relationships between markers

This approach enables researchers to simultaneously examine phospho-vimentin status in relation to cell state, cycle phase, and other relevant biological parameters within the same cell or tissue section.

What are key considerations for using Phospho-VIM (S56) Antibody in cell-based ELISA assays?

Cell-based ELISA provides a high-throughput method for quantifying phospho-vimentin levels:

Assay Design and Setup:

• The Phospho-Vimentin (S56) Cell-Based Colorimetric ELISA Kit enables quantification of phospho-vimentin (S56) proteins in different cell types

• Cells are cultured directly in 96-well plates with clear bottoms

• Sample standardization is critical - cell seeding density should be optimized and consistent

• Include appropriate positive and negative controls in each plate

Key Components:

• Anti-Phospho-Vimentin (S56) antibody (typically supplied at 100X concentration)

• Anti-Vimentin antibody for normalization to total protein levels

• HRP-conjugated secondary antibody

• Crystal violet solution for normalizing to cell number

• One-step TMB substrate for colorimetric detection

Protocol Workflow:

• Culture cells in 96-well plates

• Apply treatments (if studying modulators of phosphorylation)

• Fix cells and permeabilize

• Block non-specific binding sites

• Incubate with primary antibodies (phospho-specific and total)

• Wash and incubate with HRP-conjugated secondary antibody

• Develop with TMB substrate and measure absorbance

• Stain with crystal violet for cell number normalization

Data Analysis:

• Calculate the ratio of phospho-vimentin to total vimentin

• Normalize to cell number using crystal violet staining

• Compare treatment effects using appropriate statistical tests

• Present data as fold-change relative to control conditions

This method offers advantages over traditional Western blotting:

• Higher throughput (multiple conditions tested simultaneously)

• Lower sample requirement

• Quantitative results

• Ability to normalize to both total protein and cell number

• Faster turnaround time