Product Pathways - Cytoskeletal Signaling
PathScan® Total Vimentin Sandwich ELISA Kit #7789
When ordering five or more kits, please contact us for processing time and pricing at email@example.com.
|Kit Includes||Volume||Solution Color|
|Vimentin Mouse Ab Coated Microwells||96 tests|
|Vimentin Rabbit Detection Ab||11 ml||Green|
|Anti-Rabbit 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|
|ELISA Wash Buffer (20X)||25 ml||Colorless|
|Cell Lysis Buffer (10X) #9803||15 ml||Yellowish|
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 enclosed in parentheses are predicted to react based on 100% sequence homology.
The PathScan® Total Vimentin Sandwich ELISA Kit is a solid phase sandwich enzyme-linked immunosorbent assay (ELISA) that detects endogenous levels of total vimentin protein. A Vimentin Mouse mAb has been coated onto the microwells. After incubation with cell lysates, both phospho- and nonphospho-vimentin protein is captured by the coated antibody. Following extensive washing, a Vimentin Rabbit Detection Antibody is added to detect the captured vimentin protein. Anti-rabbit 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® Total Vimentin Sandwich ELISA Kit detects endogenous levels of vimentin protein 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.
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® Total Vimentin Sandwich ELISA Kit is shown.
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 the levels of total vimentin as detected by the 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.
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).
- Eng, L.F. et al. (2000) Neurochem. Res. 25, 1439-1451.
- Goebel, H.H. et al. (1987) Acta Histochem. Suppl. 34, 81-93.
- Leader, M. et al. (1987) Histopathology 11, 63-72.
- Helfand, B.T. et al. (2004) J. Cell Sci. 117, 133-141.
- Tang, D.D. et al. (2005) Biochem. J. 388, 773-783.
- Fomina, I.G. et al. (1990) Klin. Med. (Mosk.) 68, 125-127.
- Nieminen, M. et al. (2006) Nat. Cell Biol. 8, 156-162.
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- 7795 PathScan® Phospho-Vimentin (Ser56) Sandwich ELISA Kit
- 5741 Vimentin (D21H3) XP® Rabbit mAb
- 7074 Anti-rabbit IgG, HRP-linked Antibody
- 9807 Paclitaxel
- 9803 Cell Lysis Buffer (10X)
- 7004 TMB Substrate
- 7002 STOP Solution
For Research Use Only. Not For Use In Diagnostic Procedures.