Figure 1. Ezh2 protein was detected from COS-7, MCF7, and SK-BR-3 cells using the PathScan® Total Ezh2 Sandwich ELISA Kit #13278. The absorbance readings at 450 nm are shown in the top figure while the corresponding western blot using Ezh2 (AC22) Mouse mAb #3147 is shown in the bottom figure.
Figure 2. The relationship between protein concentration of lysates from COS-7 cells and the absorbance at 450 nm as detected by the PathScan® Total Ezh2 Sandwich ELISA Kit is shown.
|Product Includes||Volume||Solution Color|
|Ezh2 Rabbit mAb Coated Microwells||96 tests|
|Ezh2 Mouse Detection mAb||1 ea||Green (Lyophilized)|
|Anti-mouse IgG, HRP-linked Antibody (ELISA Formulated)||1 ea||Red (Lyophilized)|
|Detection Antibody Diluent||11 ml||Green|
|HRP Diluent||11 ml||Red|
|TMB Substrate 7004||11 ml|
|STOP Solution 7002||11 ml|
|Sealing Tape||2 ea|
|ELISA Wash Buffer (20X) 9801||25 ml|
|ELISA Sample Diluent||25 ml||Blue|
|Cell Lysis Buffer (10X) 9803||15 ml|
PathScan® Total Ezh2 Sandwich ELISA Kit is a solid phase sandwich enzyme-linked immunosorbent assay (ELISA) that detects endogenous levels of total Ezh2 protein. An Ezh2 specific antibody has been coated onto the microwells. After incubation with cell lysates, Ezh2 protein is captured by the coated antibody. Following extensive washing, an Ezh2 mouse mAb is added to detect the captured Ezh2 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 total Ezh2 protein.
Antibodies in kit are custom formulations specific to kit.
NOTE: Prepare solutions with purified water.
*NOTE: Some PathScan® ELISA Kits may include HRP-Linked Streptavidin in place of HRP-Linked Antibody.
NOTE: Initial color of positive reaction is blue, which changes to yellow upon addition of STOP Solution.
posted November 2013
Protocol Id: 204
PathScan® Total Ezh2 Sandwich ELISA Kit recognizes endogenous levels of Ezh2 protein as shown in Figure 1. The 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.
The polycomb group (PcG) proteins are involved in maintaining the silenced state of several developmentally regulated genes and contribute to the maintenance of cell identity, cell cycle regulation, and oncogenesis (1,2). Enhancer of zeste homolog 2 (Ezh2), a member of this large protein family, contains four conserved regions including domain I, domain II, and a cysteine-rich amino acid stretch that precedes the carboxy-terminal SET domain (3). The SET domain has been linked with histone methyltransferase (HMTase) activity. Moreover, mammalian Ezh2 is a member of a histone deacetylase complex that functions in gene silencing, acting at the level of chromatin structure (4). Ezh2 complexes methylate histone H3 at Lys9 and 27 in vitro, which is thought to be involved in targeting transcriptional regulators to specific loci (5). Ezh2 is deregulated in various tumor types, and its role, both as a primary effector and as a mediator of tumorigenesis, has become a subject of increased interest (6).
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