Anti-Vimentin antibody ValidAb™

(HB7843)
Technical documents: SDS Datasheet

Product overview

Name Anti-Vimentin antibody ValidAb™
Host Goat
Clonality Polyclonal
Target Vimentin
Description

Antibody to Vimentin - class III intermediate filament expressed in mesenchymal cells used as a marker of epithelial-mesenchymal transition. Part of the ValidAb™ range of highly validated, data-rich antibodies. 

Write Your Own Review
You're reviewing:Anti-Vimentin antibody ValidAb™
Rate this item:

Validation data

Figure 1. Vimentin and GFAP co-expression within astrocytes found within cultured neurons.

Glia in the CNS have a mesenchymal origin therefore co-express vimentin (labelled with HB7843) and GFAP (labelled with HB8267). Method: neurones were cultured from PND2 rats following established protocols (Brewer and Torricelli, 2007. Nat Protoc 2, 1490–1498) and fixed with 4% PFA on DIV21. Cells were permeabilised with 0.1% Triton X-100 before being blocked in 1% BSA, 300mM glycine. Cells were incubated overnight at 4°C in HB7843 (2µg/ml, 1:500) and HB8267 (1µg/ml, 1:1000). Secondary antibodies were incubated for 1 hour at RT (donkey polyclonal anti-mouse Dy488, Thermofisher SA5-10166, 1:300 and donkey polyclonal anti-goat Dy594, Thermofisher SA5-10088, 1:300) before nuclei were counterstained using DAPI (HB0747, 1µg/ml). For more detail please see our ICC protocol. Images were captured using a Leica SPE confocal laser scanning microscope coupled to a Leica DMi8 inverted epifluorescence microscope. The image was captured in Lightning deconvolution mode using a 63x objective with 0.30µm z-spacing, 405nm (11.8% power @ 851V gain (PMT)), 496nm (0.5% power @ 17.7% gain (Hyd)) and 561nm (2.0% power @ 14.7% gain (HyD)) lasers. Following capture the stack was flattened using a maximum Z projection in ImageJ (Schindelin et al., 2012. Nat Methods, 9(7), 676–682).

Figure 2. Independent antibody validation of HB7843 in glia within a cultured rat neurone preparation

Both HB6497 (mouse monoclonal) and HB7843 (goat polyclonal) immunoreactivity within mixed neuronal cultures overlaps therefore providing evidence for antibody specificity. Method: neurones were cultured from PND2 rats following established protocols (Brewer and Torricelli, 2007. Nat Protoc 2, 1490–1498) and fixed with 4% PFA on DIV21. Cells were permeabilised with 0.1% Triton X-100 before being blocked in 1% BSA, 300mM glycine. Cells were incubated overnight at 4°C in HB6497 (1µg/ml, 1:1000) and HB7843 (2µg/ml, 1:500). Secondary antibodies were incubated for 1 hour at RT (donkey polyclonal anti-mouse Dy488, Thermofisher SA5-10166, 1:300 and donkey polyclonal anti-goat Dy594, Thermofisher SA5-10088, 1:300) before nuclei were counterstained using DAPI (HB0747, 1µg/ml). For more detail please see our ICC protocol. Images were captured using a Leica SPE confocal laser scanning microscope coupled to a Leica DMi8 inverted epifluorescence microscope. The image was captured in Lightning deconvolution mode using a 63x objective with 0.27µm z-spacing, 405nm (11.8% power @ 825V gain (PMT)), 561nm (1.0% power @ 10.8% gain (Hyd)) and 496nm (0.5% power @ 27.1% gain (HyD)) lasers. Following capture the stack was flattened using a maximum Z projection in ImageJ (Schindelin et al., 2012. Nat Methods, 9(7), 676–682).

Figure 3. Independent antibody validation of HB7843 in glia within a cultured rat neurone preparation

Both HB6497 (mouse monoclonal) and HB7843 (goat polyclonal) immunoreactivity within mixed neuronal cultures overlaps therefore providing evidence for antibody specificity. Method: neurones were cultured from PND2 rats following established protocols (Brewer and Torricelli, 2007. Nat Protoc 2, 1490–1498) and fixed with 4% PFA on DIV21. Cells were permeabilised with 0.1% Triton X-100 before being blocked in 1% BSA, 300mM glycine. Cells were incubated overnight at 4°C in HB6497 (1µg/ml, 1:1000) and HB7843 (2µg/ml, 1:500). Secondary antibodies were incubated for 1 hour at RT (donkey polyclonal anti-mouse Dy488, Thermofisher SA5-10166, 1:300 and donkey polyclonal anti-goat Dy594, Thermofisher SA5-10088, 1:300) before nuclei were counterstained using DAPI (HB0747, 1µg/ml). For more detail please see our ICC protocol. Images were captured using a Leica SPE confocal laser scanning microscope coupled to a Leica DMi8 inverted epifluorescence microscope. The image was captured in Lightning deconvolution mode using a 63x objective with 0.27µm z-spacing, 405nm (11.8% power @ 825V gain (PMT)), 561nm (1.0% power @ 10.8% gain (Hyd)) and 496nm (0.5% power @ 27.1% gain (HyD)) lasers. Following capture the stack was flattened using a maximum Z projection in ImageJ (Schindelin et al., 2012. Nat Methods, 9(7), 676–682).

Figure 4. Vimentin and GFAP expression in a rat cultured neuron preparation visualised using HB7843 and HB8267

Vimentin and GFAP are both expressed in glial cells and can be seen to express at different levels in individual cells creating a colour gradient across the cell population. Method: neurones were cultured from PND2 rats following established protocols (Brewer and Torricelli, 2007. Nat Protoc 2, 1490–1498) and fixed with 4% PFA on DIV21. Cells were permeabilised with 0.1% Triton X-100 followed by blocking in 1% BSA, 300mM glycine. HB8267 was incubated overnight (4°C) at a 1:1000 dilution (1µg/ml) with HB7843 being incubated at a 1:500 dilution (2µg/ml). Secondary antibodies (Polyclonal donkey anti-mouse DyLight 488 conjugated, Thermofisher SA5-10166, 1:300 dilution and Polyclonal donkey anti-goat DyLight 594 conjugated, Thermofisher SA5-10088, 1:300 dilution) were incubated for 1 hour at room temperature. Nuclei were visualised using 1µg/ml DAPI (HB0747). For more detail please see our ICC protocol. Images were captured using a Leica SP8 AOBS confocal laser scanning microscope attached to a Leica DMi8 inverted epifluorescence microscope. The image was captured in a tile scan and z-stack (0.49µm spacing) using Lightning adaptive deconvolution and a 63x objective. Lasers used were 405nm (20.0% power, PMT: 712.7V gain), 496nm (1.0% power, HyD: 14.1% gain) and 561nm (1.5% power, HyD: 10% gain). The tile scan was merged using LASX before the stack was flattened using a maximum Z projection in ImageJ (Schindelin et al., 2012. Nat Methods, 9(7), 676–682).

Figure 5. Concentration response of HB7843 in mixed neuronal cultures.

HB7843 optimally reveals the dense network of vimentin containing intermediate filaments at 2µg/ml (1:500 dilution). Method: neurones were cultured from PND2 rats following established protocols (Brewer and Torricelli, 2007. Nat Protoc 2, 1490–1498) and fixed with 4% PFA on DIV21. Cells were permeabilised with 0.1% Triton X-100 before being blocked in 1% BSA, 300mM glycine. Cells were incubated overnight at 4°C in HB7843 (concentrations ranging from 2µg/ml(1:500) to 0.25µg/ml (1:4000)) before being incubated in secondary antibody (donkey polyclonal anti-goat Dy594, Thermofisher SA5-10088, 1:300) for 1 hour at RT. Nuclei were counterstained using DAPI (HB0747, 1µg/ml). For more detail please see our ICC protocol. Images were captured using a Leica SPE confocal laser scanning microscope coupled to a Leica DMi8 inverted epifluorescence microscope. Images were captured in Lightning deconvolution mode using a 63x objective with the following exposure settings:

  • 1:500, 405nm: 11.8% power, 851V gain (PMT); 496nm: 0.5% power, 17.7% gain (HyD); 561nm: 2.0% power, 14.7% gain (HyD),
  • 1:1000, 405nm: 11.8% power, 851V gain (PMT); 496nm: 0.5% power, 24.9% gain (HyD); 561nm: 2.0% power, 26.7% gain (HyD),
  • 1:2000, 405nm: 11.8% power, 851V gain (PMT); 496nm: 1.0% power, 32.0% gain (HyD); 561nm: 5.0% power, 22.1% gain (HyD),
  • 1:4000, 405nm: 11.8% power, 851V gain (PMT); 496nm: 1.0% power, 10.0% gain (HyD); 561nm: 5.0% power, 42.9% gain (HyD),

Following capture the stacks were flattened using a maximum Z projection in ImageJ (Schindelin et al., 2012. Nat Methods, 9(7), 676–682).

Figure 6. Vimentin expression in HeLa cells visualised using HB7843.

HB7843 reveals the network of intermediate filaments present within cultured HeLa cells. Method: HeLa cells were cultured in 10% FBS, 1% pen/strep in DMEM before being plated onto PDL coated coverslips and incubated for 2 days. Cells were then fixed in 4% PFA before being permeabilised with 0.1% Triton X-100. Cells were blocked in 1% BSA, 300mM glycine then HB7843 was incubated at 4°C overnight (1:500, 2µg/ml). Following washing, cells were incubated in a polyclonal donkey anti-goat DyLight 594 conjugated secondary antibody (1:300, Thermofisher SA5-10088) for 1 hour at room temperature. Nuclei were stained using 1µg/ml DAPI (HB0747). For more detail please see our ICC protocol. The image was captured using a Leica SP8 AOBS confocal laser scanning microscope attached to a Leica DMi8 inverted epifluorescence microscope. The image was captured in a z-stack (0.37µm spacing) using Lightning adaptive deconvolution and a 63x objective. Lasers used were 405nm (3.2% power, PMT: 542.1V gain) and 561nm (1.0% power, HyD: 28.6% gain). Following capture, the stack was flattened using a maximum Z projection in ImageJ (Schindelin et al., 2012. Nat Methods, 9(7), 676–682).

Figure 7. HB7843 immunoreactivity in a range of cell and tissue lysates

HB7843 revealed a band corresponding with Vimentin at 55.6kDa in human cell line samples and brain cytosol preparations from mouse and rat. There was much immunoreactivity in liver samples. Method: mouse brain and rat brain cytosol fractions were prepared following previous work (Molnar et al., 1993. Neuroscience 53:307-326) from freshly collected adult brains. Other tissue lysates were prepared following established protocols from freshly dissected tissue (see our guide on WB sample preparation). HEK293 and HeLa cells were cultured in 10% FBS, 1% pen/strep in DMEM before having proteins extracted. Samples were loaded (20µg / lane) onto a 12% acrylamide gel alongside a protein ladder (Biorad precision plus dual colour, 1610374) before being run at 60V for 45 minutes followed by 120V for 90 minutes. Wet transfer to a PVDF membrane was completed in 90 minutes using 400mA. The membrane was blocked for 1hr in 5% non-fat dry milk before being incubated overnight at 4°C in HB7843 at a 1:1,000 dilution (1µg/ml). Following washing, the membrane was incubated in secondary antibody (1:10,000 dilution, Polyclonal donkey anti-goat HRP conjugated, Thermofisher 77-56-060520) for 2hrs. For more detail please see our Western blotting protocol. Detection was accomplished using Clarity Western ECL substrate (BioRad, 1705061) and a Licor Odyssey Fc imaging system (ECL channel: 10 min exposure, 700nm channel: 30 sec exposure). Following imaging, the membrane was stripped with two changes of stripping buffer (HB7756) before being washed, blocked for 30 minutes in 1% fish skin gelatin and incubated in HB9177 (1:4,000 dilution, 0.25µg/ml) for 3 days at 4°C. Following washing, the membrane was incubated in secondary antibody (1:10,000 dilution, Polyclonal goat anti-mouse HRP conjugated, Sigma Aldrich A3682) for 2hrs and visualised again using Clarity Western ECL substrate (BioRad, 1705061) and a Licor Odyssey Fc imaging system (ECL channel: 2 min exposure, 700nm channel: 30 sec exposure).

Product information

Immunogen

Recombinant human vimentin expressed in and purified from E. coli

Purification

Affinity purification using immunogen as ligand

Concentration 1 mg/ml
Formulation 50% PBS, 50% glycerol plus 5mM sodium azide
Predicted species reactivity Mouse, Rat, Human, Pig, Horse, Cow, Monkey
Tested species reactivity Rat, Human

Tested applications

Applications ICC, WB
Western blot optimal concentration

1µg/ml (1:1000 dilution) as tested in HEK293T and HeLa cell lysate. Please not that while this antibody reacts with a high signal to noise ratio in human derived cell line lysate this ratio is much lower in mouse and rat tissue lysates.

ICC optimal concentration

2µg/ml (1:500 dilution) as tested in primary mixed rat neuronal cultures.

Positive control

Vimentin is highly expressed in human cell lines such as HEK293 and HeLa while also being expressed at high levels in glia within the CNS.

Negative control

Vimentin is not expressed in some human derived cell lines such as HepG2 and RT4 cells while in tissue samples vimentin is not expressed in hepatocytes but is in other cell types within the liver.

Open data link

Please follow this link to OSF

Target information

UniProt ID P08670
Structure image  Chemical Structure
Gene name VIM
NCBI full gene name VIM – Vimentin
Entrez gene ID

7431

Amino acids

466 - 53.65kDa

Isoforms

Vimentin has no fully described isoforms.

Expression

Vimentin is expressed in tissues with a mesenchymal origin including glia, fibroblasts, endothelial cells lining blood vessels, renal tubular cells and many cells of the immune system amongst others. Vimentin is also expressed in cells undergoing a epithelial-mesenchymal transition therefore used as a marker for this.

Subcellular expression

Vimentin is expressed in the intermediate filaments of the cytoskeleton.

Processing

The initiator methionine is removed to form the mature protein.

Post translational modifications

Subject to phosphorylation on multiple residues alongside posessing sumoylation, N-6 acetylation and N-6 succinylation sites.

Homology (compared to human)

Mouse and rat show 97.4% identity to human Vimentin in a BLAST search.

Similar proteins

The following proteins were identified as being similar to Vimentin in a BLAST search:

  • Desmin - 62.9% identity
  • GFAP - 58.1% identity
  • Peripherin - 57.1% identity

Storage & Handling

Storage instructions

-20°C

Important This product is for RESEARCH USE ONLY and is not intended for therapeutic or diagnostic use. Not for human or veterinary use

FAQs

What guarantee do you have that my Vimentin antibody will perform as expected?

We guarantee that your Vimentin antibody will work for the applications and species we list on the datasheet. If the antibody fails to perform as expected then we are happy to offer a 100% refund guarantee. For more details please see our guarantee policy.

What protocols are available for use with this Vimentin antibody?

We have made a comprehensive collection of protocols that we have used in our experiments to validate this Vimentin antibody.

What counterstains do you recommend for use in ICC and IHC with this Vimentin antibody?

We recommend using either DAPI, Hoechst 33342 or Propidium Iodide to label cell nuclei. In some experiments it is also helpful to label actin filaments in the cytoskeleton using a Phalloidin conjugate such as FITC Phalloidin or Rhodamine Phalloidin-TRITC.

Will my Vimentin antibody work against species that have not been listed on the datasheet?

A species not being listed doesn’t mean that the Vimentin antibody won’t work, just that we haven’t tested it. If you test one of our antibodies in a new species please let us know (positive or negative)!

Any other questions?

For any other questions about our antibody products please see our technical FAQs for antibodies

References for Anti-Vimentin antibody ValidAb™

References are publications that support the biological activity of the product