Overview

  • Product name
    Anti-Vimentin (phospho S55) antibody [4A4]
    See all Vimentin primary antibodies
  • Description
    Mouse monoclonal [4A4] to Vimentin (phospho S55)
  • Host species
    Mouse
  • Specificity
    Does not detect non-phosphorylated Vimentin or Vimentin phosphorylated by camp dependant kinase, protein kinase C or Ca2+ calmodulin dependant kinase II.
  • Tested applications
    Suitable for: WB, ELISA, ICC/IF, IHC-P, IHC-Fr, Flow Cytmore details
  • Species reactivity
    Reacts with: Mouse, Rat, Human, Ferret
  • Immunogen

    Synthetic peptide:

    SLYSSSPGGAYV

    corresponding to amino acids 50 - 61 of Mouse Vimentin, containing phosphorlyated serine 55, and conjugated to KLH.

  • Positive control
    • The antibody was tested positive with the following cell lines via WB: U251, NIH-3T3, and 3Y1-B.

Properties

Applications

Our Abpromise guarantee covers the use of ab22651 in the following tested applications.

The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.

Application Abreviews Notes
WB Use a concentration of 1 - 5 µg/ml. Detects a band of approximately 57 kDa (predicted molecular weight: 54 kDa).
ELISA Use at an assay dependent concentration.
ICC/IF Use a concentration of 1 µg/ml.
IHC-P Use at an assay dependent concentration. PubMed: 20436478
IHC-Fr Use at an assay dependent concentration. PubMed: 20436478
Flow Cyt Use 1µg for 106 cells.

ab170192 - Mouse monoclonal IgG2b, is suitable for use as an isotype control with this antibody.

Target

  • Function
    Vimentins are class-III intermediate filaments found in various non-epithelial cells, especially mesenchymal cells. Vimentin is attached to the nucleus, endoplasmic reticulum, and mitochondria, either laterally or terminally.
    Involved with LARP6 in the stabilization of type I collagen mRNAs for CO1A1 and CO1A2.
  • Tissue specificity
    Highly expressed in fibroblasts, some expression in T- and B-lymphocytes, and little or no expression in Burkitt's lymphoma cell lines. Expressed in many hormone-independent mammary carcinoma cell lines.
  • Involvement in disease
    Cataract 30
  • Sequence similarities
    Belongs to the intermediate filament family.
  • Domain
    The central alpha-helical coiled-coil rod region mediates elementary homodimerization.
    The [IL]-x-C-x-x-[DE] motif is a proposed target motif for cysteine S-nitrosylation mediated by the iNOS-S100A8/A9 transnitrosylase complex.
  • Post-translational
    modifications
    Filament disassembly during mitosis is promoted by phosphorylation at Ser-55 as well as by nestin (By similarity). One of the most prominent phosphoproteins in various cells of mesenchymal origin. Phosphorylation is enhanced during cell division, at which time vimentin filaments are significantly reorganized. Phosphorylation by PKN1 inhibits the formation of filaments. Phosphorylated at Ser-56 by CDK5 during neutrophil secretion in the cytoplasm. Phosphorylated by STK33.
    O-glycosylated during cytokinesis at sites identical or close to phosphorylation sites, this interferes with the phosphorylation status.
    S-nitrosylation is induced by interferon-gamma and oxidatively-modified low-densitity lipoprotein (LDL(ox)) possibly implicating the iNOS-S100A8/9 transnitrosylase complex.
  • Cellular localization
    Cytoplasm.
  • Information by UniProt
  • Database links
  • Form
    Vimentin is found in connective tissue and in the cytoskeleton.
  • Alternative names
    • CTRCT30 antibody
    • Epididymis luminal protein 113 antibody
    • FLJ36605 antibody
    • HEL113 antibody
    • VIM antibody
    • VIME_HUMAN antibody
    • Vimentin antibody
    see all

Images

  • ab22651 staining Vimentin in Ferret brain tissue sections by Immunohistochemistry (IHC-Fr - frozen sections). Tissue was fixed with paraformaldehyde, permeabilized with Triton-X and blocked with 7.5ug/ml 0.1 M glycine buffer for 30 minutes at room temperature. Samples were incubated with primary antibody (1/50) for 16 hours at 4°C. A Cy2®-conjugated Goat anti-rabbit IgG polyclonal (1/500) was used as the secondary antibody.

    See Abreview

  • All lanes :

    Lane 1 : U251 interphase cells
    Lane 2 : U251 M phase cells

    Lysates/proteins at 5 µg/ml per lane.

    Predicted band size: 54 kDa



    Western Blot analysis using ab22651

  • ab22651 at 1µg/ml staining Vimentin (phospho S55) in human U251 cells by Immunocytochemistry/ Immunofluorescence.

  • Overlay histogram showing HeLa cells stained with ab22651 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab22651, 1µg/1x106 cells) for 30 min at 22ºC. The secondary antibody used was DyLight® 488 goat anti-mouse IgG (H+L) (ab96879) at 1/500 dilution for 30 min at 22ºC. Isotype control antibody (black line) was mouse IgG2b [PLPV219] (ab91366, 2µg/1x106 cells) used under the same conditions. Acquisition of >5,000 events was performed. This antibody gave a positive signal in HeLa cells fixed with 4% paraformaldehyde (10 min)/permeabilized with 0.1% PBS-Tween for 20 min used under the same conditions.

References

This product has been referenced in:
  • Boucherie C  et al. Neural progenitor fate decision defects, cortical hypoplasia and behavioral impairment in Celsr1-deficient mice. Mol Psychiatry 23:723-734 (2018). IHC (PFA fixed) ; Mouse . Read more (PubMed: 29257130) »
  • Römer S  et al. Neural Progenitors in the Developing Neocortex of the Northern Tree Shrew (Tupaia belangeri) Show a Closer Relationship to Gyrencephalic Primates Than to Lissencephalic Rodents. Front Neuroanat 12:29 (2018). Read more (PubMed: 29725291) »
See all 17 Publications for this product

Customer reviews and Q&As

1-4 of 4 Abreviews or Q&A

Application
Immunocytochemistry/ Immunofluorescence
Sample
Human Cell (Colon carcinoma)
Specification
Colon carcinoma
Fixative
Paraformaldehyde
Permeabilization
Yes - 0.5% Triton in PBS
Blocking step
BSA as blocking agent for 30 minute(s) · Concentration: 1% · Temperature: 37°C

Dr. Eleni Petsalaki

Verified customer

Submitted Dec 04 2012

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Immunohistochemistry (Frozen sections)
Sample
Ferret Tissue sections (Brain)
Specification
Brain
Fixative
Paraformaldehyde
Permeabilization
Yes - Triton-X
Blocking step
0.1 M glycine buffer as blocking agent for 30 minute(s) · Concentration: 7.5µg/mL · Temperature: RT°C

Abcam user community

Verified customer

Submitted Aug 31 2010

Question
Answer

Mouse vimentin is Ser-55, which is Ser-56 in human vimentin. The antibody will recognize either (the immunogen is from mouse vimentin so the mouse phosphorylation site was chosen for the product name/description).

Read More

Answer

Thank you for your phone call. This antibody was tested positive with the following cell lines via WB: U251, NIH-3T3, and 3Y1-B. I would recommend using one of these as a positive control. It also tests positive with the phospho-Vimentin (Ser55) peptide and reacts negative with non-phospho-Vimentin, and Vimentin phosphorylated by cAMP-dependent kinase, protein kinase C, or calclium-calmodulin-dependent protein kinase II. Below is the WB protocol. Please contact us again if you have any additional questions. 1.Determine protein concentration using an assay such as Lowry or Bradford. 2.Boil protein samples in the smallest possible volume of Laemmli sample buffer for 5 minutes immediately after harvesting (Laemmli, 1 970). Some antigens are heat labile and heating at 70C is recommended. 3.Load protein samples and controls. Determine appropriate protein by running preliminary gels and staining with Coomassie Blue. Include unstained or pre-stained molecular weight markers. Pre-stained molecular weight markers will verify that the gel has run properly. 4.Start electrophoresis at 100V. After the dye front has moved into the separating gel, increase to 200V. Stop electrophoresis when the dye front reaches the bottom of the gel. Protein Transfer 1.Transfer separated protein bands onto a suitable membrane, such as nitrocellulose paper or PVDF (for small MW proteins). 2.In semi-dry transfer , the gel and membrane are equilibrated in transfer buffer for 30 minutes prior to transfer. Semi-dry transfer is run for 30 minutes at 20V at room temperature. 3.Equilibration of the gel and membrane is not necessary when using a submerged transfer. With a submerged-type apparatus transfer at 4C for 1 hour at 100V. For submerged transfers, use 1X Towbin Buffer: 1X Towbin Buffer = 25mM Tris + 192mM Glycine + 20% methanol 4.Assemble the apparatus according to manufacturers’ instructions. 5.With either method, at no time should the apparatus or buffer be allowed to become hot. Use a cooling coil or run the transfer in a cold room to avoid the generation of gas bubbles in the sandwich. 6.Verify protein transfer by staining the membrane with Ponceau S stain (0.1% Ponceau S in 5% acetic acid Sigma P 7170). Incubate for ~30 seconds. Mark the MW standards lane with a pencil if pre-stained markers were not used. Destain immediately in PBS/0.05% Tween 20 using agitation. The staining is reversible, and may be repeated if necessary. Blocking 1.Block non-specific binding sites on membrane. Our preferred blocking solution is 5% Blotto: 5% wt/vol nonfat dry milk/0.05% Tween 20/0.02% NaN3 in PBS. Note: thimersol is used in place of azide for immunoblotting using ECL method of color development. The membrane is sealed in a bag containing the solution with as few air bubbles as possible and incubated overnight at room temperature on the rotator. All blocking solutions should be stored at 4?C. Addition of Antibody 1.Incubate the membrane with the primary antibody for 1 hour at room temperature at appropriate dilution in 5% Blotto. Use suggested working dilution stated in Technical Specifications sheet as a starting point for your assay. You may empirically determine a more appropriate dilution, which is dependant on the individual experiment and sample source. If you are using a more sensitive detection system, such as ECL, it may be necessary to use a more dilute preparation. 2.Wash with PBS0.05 Tween 20 three times for 5 minutes. Wash at room temperature with moderate agitation. 3.Incubate the membrane with the second antibody conjugated to the appropriate detection substrate (i.e. AP) for 1 hour at room temperature, as in Step 1. Secondary antibodies from Stressgen are generally used at a dilution of 1:1000 in 5% Blotto. 4.Wash three times with PBS/0.05% Tween 20 for 5 minutes per wash.

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