Cell Signaling Technology

Product Pathways - Cytoskeletal Signaling

PathScan® Phospho-Vimentin (Ser56) Sandwich ELISA Kit #7795

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Kit Includes Volume Solution Color
P-Vimentin(Ser56) Rabbit Ab Coated Microwells 96 assays
Vimentin Mouse Detection Ab 11 ml Green
Anti-Mouse IgG, HRP-linked Antibody 11 ml Red
TMB Substrate #7004 11 ml Colorless
STOP Solution #7002 11 ml Colorless
Sealing Tape 2 sheets
ELISA Sample Diluent 25 ml Blue
Cell Lysis Buffer (10X) #9803 15 ml Yellowish
ELISA Wash Buffer (20X) 25 ml Colorless

Note: 12 8-well modules –Each module is designed to break apart for 8 tests.
Note: Kit should be stored at 4°C with the exception of Cell Lysis Buffer, which is stored at –20°C (packaged separately).

Species Cross-Reactivity

H

Reactivity Key:  H=Human
Species enclosed in parentheses are predicted to react based on 100% sequence homology.

Description

The PathScan® Phospho-Vimentin (Ser56) Sandwich ELISA Kit is a solid phase sandwich enzyme-linked immunosorbent assay (ELISA) that detects endogenous levels of vimentin protein when phosphorylated at Ser56. A Phospho-Vimentin (Ser56) Rabbit Antibody has been coated onto the microwells. After incubation with cell lysates, phospho-vimentin protein is captured by the coated antibody. Following extensive washing, a Vimentin Mouse Detection Antibody is added to detect the captured phospho-vimentin protein. Anti-mouse IgG, HRP-linked antibody is then used to recognize the bound detection antibody. HRP substrate, TMB, is added to develop color. The magnitude of optical density for this developed color is proportional to the quantity of vimentin protein.Antibodies in kit are custom formulations specific to kit.

Specificity / Sensitivity

The PathScan® Phospho-Vimentin (Ser56) Sandwich ELISA Kit detects endogenous levels of vimentin protein when phosphorylated at Ser56 as shown in Figure 1. Kit sensitivity is shown in Figure 2. This kit detects proteins from the indicated species, as determined through in-house testing, but may also detect homologous proteins from other species.

ELISA

ELISA

Figure 2. The relationship between the protein concentration of lysates from HeLa cells, treated with Paclitaxel #9807 or hydroxyurea, and the absorbance at 450 nm using the PathScan® Phospho-Vimentin (Ser56) Sandwich ELISA Kit is shown.

ELISA - Western correlation

ELISA - Western correlation

Figure 1. Treatment of HeLa cells with Paclitaxel stimulates phosphorylation of vimentin at Ser56, while treatment with hydroxyurea reduces that phosphorylation, as detected by the PathScan® Phospho-Vimentin (Ser56) Sandwich ELISA Kit #7795. Neither treatment affects levels of total vimentin as detected by PathScan® Total Vimentin Sandwich ELISA Kit #7789. HeLa cells (80-90% confluent) were treated with 100 nM Paclitaxel #9807 or 4 mM hydroxyurea for 20 hr and lysed. The absorbance readings at 450 nm are shown in the top figure, while the corresponding western blots using Vimentin (D21H3) XP® Rabbit mAb #5741 (left panel) or Phospho-Vimentin (Ser56) (D5H2) Rabbit mAb #7391 (right panel) are shown in the bottom figure.

Background

The cytoskeleton consists of three types of cytosolic fibers: microfilaments (actin filaments), intermediate filaments, and microtubules. Major types of intermediate filaments are distinguished by their cell-specific expression: cytokeratins (epithelial cells), glial fibrillary acidic protein (GFAP) (glial cells), desmin (skeletal, visceral, and certain vascular smooth muscle cells), vimentin (mesenchyme origin), and neurofilaments (neurons). GFAP and vimentin form intermediate filaments in astroglial cells and modulate their motility and shape (1). In particular, vimentin filaments are present at early developmental stages, while GFAP filaments are characteristic of differentiated and mature brain astrocytes. Thus, GFAP is commonly used as a marker for intracranial and intraspinal tumors arising from astrocytes (2). Research studies have shown that vimentin is present in sarcomas, but not carcinomas, and its expression is examined in conjunction with that of other markers to distinguish between the two (3). Vimentin's dynamic structural changes and spatial re-organization in response to extracellular stimuli help to coordinate various signaling pathways (4). Phosphorylation of vimentin at Ser56 in smooth muscle cells regulates the structural arrangement of vimentin filaments in response to serotonin (5,6). Remodeling of vimentin and other intermediate filaments is important during lymphocyte adhesion and migration through the endothelium (7).

  1. Eng, L.F. et al. (2000) Neurochem. Res. 25, 1439-1451.
  2. Goebel, H.H. et al. (1987) Acta Histochem. Suppl. 34, 81-93.
  3. Leader, M. et al. (1987) Histopathology 11, 63-72.
  4. Helfand, B.T. et al. (2004) J. Cell Sci. 117, 133-141.
  5. Tang, D.D. et al. (2005) Biochem. J. 388, 773-783.
  6. Fomina, I.G. et al. (1990) Klin. Med. (Mosk.) 68, 125-127.
  7. Nieminen, M. et al. (2006) Nat. Cell Biol. 8, 156-162.

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