FastScan™ Phospho-YB1 (Ser102) ELISA Kit #42976
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Supporting Data
| REACTIVITY | H M |
Species Cross-Reactivity Key:
- H-Human
- M-Mouse
Product Information
Product Description
The FastScan™ Phospho-YB1 (Ser102) ELISA Kit is a sandwich enzyme-linked immunosorbent assay (ELISA) that detects endogenous levels of YB1 when phosphorylated at Ser102. 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 phospho-YB1 (Ser102) 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 phospho-YB1 (Ser102).
*Antibodies in kit are custom formulations specific to kit.
IMPORTANT: This FastScan™ ELISA Kit requires 4 washes at Step 6 of the protocol.
Protocol
Specificity / Sensitivity
The FastScan™ Phospho-YB1 (Ser102) ELISA Kit detects endogenous levels of YB1 when phosphorylated at Ser102, 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:
Human, Mouse
Background
The Y-box binding protein 1 (YB1) belongs to a family of evolutionarily conserved, multifunctional Y-box proteins that bind single-stranded DNA and RNA and function as regulators of transcription, RNA metabolism, and protein synthesis (1). YB1 binds to Y-box sequences (TAACC) found in multiple gene promoters and can positively or negatively regulate transcription. YB1 activates genes associated with proliferation and cancer, such as cyclin A, cyclin B1, matrix metalloproteinase-2 (MMP-2), and the multi-drug resistance 1 (MDR1) gene (2-4). YB1 represses genes associated with cell death, including the Fas cell death-associated receptor and the p53 tumor suppressor gene (5-7). It also interacts with the RNA-splicing factor SRp30c and stabilizes interleukin-2 (IL-2) mRNA upon induction of T lymphocytes by IL-2 (8,9). The majority of YB1 protein localizes to the cytoplasm, with a minor pool found in the nucleus; however, nuclear localization appears to be critical for its role in promoting proliferation. Nuclear translocation is cell cycle regulated, with YB1 protein accumulating in the nucleus during G1/S phase (2). In addition, nuclear translocation is induced in response to extracellular stimuli such as hyperthermia and UV irradiation, or treatment of cells with thrombin, interferons, or insulin-like growth factor (IGF-I) (2,10). Treatment of the MCF7 breast cancer cell line with IGF-I results in Akt-mediated phosphorylation of YB1 at Ser102, which is required for nuclear translocation of YB1 and its ability to promote anchorage-independent growth (10). Research studies have shown that YB1 is overexpressed in many malignant tissues, including breast cancer, non-small cell lung carcinoma, ovarian adenocarcinomas, human osteosarcomas, colorectal carcinomas, and malignant melanomas. Investigators have shown that nuclear YB1 expression correlates with high levels of proliferation, drug resistance, and poor tumor prognosis (2,7,10).
- Matsumoto, K. and Wolffe, A.P. (1998) Trends Cell Biol. 8, 318-23.
- Jurchott, K. et al. (2003) J. Biol. Chem. 278, 27988-96.
- Mertens, P.R. et al. (1997) J. Biol. Chem. 272, 22905-12.
- Uchiumi, T. et al. (1993) Cell Growth Differ. 4, 147-57.
- Lasham, A. et al. (2000) Gene 252, 1-13.
- Lasham, A. et al. (2003) J. Biol. Chem. 278, 35516-23.
- Homer, C. et al. (2005) Oncogene 24, 8314-25.
- Raffetseder, U. et al. (2003) J. Biol. Chem. 278, 18241-8.
- Chen, C.Y. et al. (2000) Genes Dev. 14, 1236-48.
- Sutherland, B.W. et al. (2005) Oncogene 24, 4281-92.
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