Figure 1. The relationship between protein concentration of lysates from HeLa cells and MCF7 cells and the absorbance at 450 nm as detected by the FastScan™ Total Vimentin ELISA Kit #87105 is shown. HeLa and MCF7 cells were grown to 80-90% confluence at 37°C and then lysed.
Figure 2. Vimentin protein is expressed in a variety of human cell lines but absent in others, as detected by using the FastScan™ Total Vimentin ELISA Kit #87105. The absorbance readings at 450 nm are shown in the upper figure, while the corresponding western blots using vimentin and β-actin antibodies are shown in the bottom figure.
|Product Includes||Volume (with Count)||Solution Color|
|FastScan™ ELISA Microwell Strip Plate, 96 Well 53257||1 x 96 tests|
|Vimentin Rabbit Capture mAb||1 x 1 ea||Green (Lyophilized)|
|Vimentin Mouse HRP-linked mAb||1 x 1 ea||Red (Lyophilized)|
|FastScan™ ELISA Capture Antibody Diluent||1 x 3 ml||Green|
|FastScan™ ELISA HRP Antibody Diluent||1 x 3 ml|
|TMB Substrate 7004||1 x 11 ml|
|STOP Solution 7002||1 x 11 ml|
|Sealing Tape||1 x 1 ea|
|ELISA Wash Buffer (20X) 9801||1 x 25 ml|
|FastScan™ ELISA Cell Extraction Buffer (5X) 69905||1 x 10 ml|
|FastScan™ ELISA Cell Extraction Enhancer Solution (50X) 25243||1 x 1 ml|
|FastScan™ ELISA Kit #87105 Positive Control||2 x 1 ea|
NOTE: Prepare solutions with deionized/purified water or equivalent.
Prepare only as much reagent as needed on the day of the experiment.
*IMPORTANT: The provided FastScan™ ELISA Cell Extraction Enhancer Solution (50X) may precipitate when stored at 4°C. To dissolve, warm briefly at 37°C and mix gently. The FastScan™ ELISA Cell Extraction Enhancer Solution (50X) can be stored at room temperature to avoid precipitation.
NOTE: The 1X Cell Extraction Buffer contains phosphatase inhibitors. Protease inhibitors should be added to the 1X Cell Extraction Buffer immediately prior to lysing cells. Additional phosphatase inhibitors can also be added (e.g. Protease/Phosphatase Inhibitor Cocktail (100X) #5872, not supplied).
For adherent cells
For suspension cells
NOTE: Equilibrate all materials and prepared reagents to room temperature prior to running the assay.
*NOTE: Certain FastScan™ ELISA Kits may require additional washes at this step. Any requirements for additional washes will be specifically noted in the product “Description” of the kit’s datasheet.
NOTE: Initial color of positive reaction is blue, which changes to yellow upon addition of STOP Solution.
posted May 2018
revised November 2018
Protocol Id: 1704
The FastScan™ Total Vimentin ELISA Kit is a sandwich enzyme-linked immunosorbent assay (ELISA) that detects endogenous levels of Vimentin. To perform the assay, sample is incubated with a capture antibody conjugated with a proprietary tag and a second detection antibody linked to HRP, forming a sandwich with Vimentin in solution. This entire complex is immobilized to the plate via an anti-tag antibody. The wells are then washed to remove unbound material. TMB is then added. The magnitude of observed signal is proportional to the quantity of Vimentin. Antibodies in kit are custom formulations specific to kit.
The FastScan™ Total Vimentin ELISA Kit detects endogenous levels of Vimentin as shown in Figure 1. This kit detects proteins from the indicated species, as determined through in-house testing, but may also detect homologous proteins from other species.Species Reactivity:
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).
During mitosis, CDK1 phosphorylates vimentin at Ser56. This phosphorylation provides a PLK binding site for vimentin-PLK interaction. PLK further phosphorylates vimentin at Ser82, which might serve as memory phosphorylation site and play a regulatory role in vimentin filament disassembly (8,9). Additionally, studies using various soft-tissue sarcoma cells have shown that phosphorylation of vimentin at Ser39 by Akt1 enhances cell migration and survival, suggesting that vimentin could be a potential target for soft-tissue sarcoma targeted therapy (10,11).
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Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
FastScan™ ELISA is a trademark of Cell Signaling Technology, Inc.
U.S. Patents 9,086,407, 9,261,500, and 9,476,874, foreign equivalents, and child patents deriving therefrom.