Cell Signaling Technology

Product Pathways - PathScan ELISA

PathScan® Phospho-YB1 (Ser102) Sandwich ELISA Kit #7249

When ordering five or more kits, please contact us for processing time and pricing at sales@cellsignal.com.

Kit Includes Volume Solution Color
YB-1 Rabbit Ab Coated Microwells 96 tests
P-YB1(Ser102) 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
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 Cross-Reactivity

H

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

Description

The PathScan® Phospho-YB1 (Ser102) Sandwich ELISA Kit is a solid phase sandwich enzyme-linked immunosorbent assay (ELISA) that detects endogenous levels of YB1 when phosphorylated at Ser102. A YB1 rabbit antibody has been coated onto the microwells. After incubation with cell lysates, YB1 protein is captured by the coated antibody. Following extensive washing, a phospho-YB1 (Ser102) mouse detection antibody is added to detect the captured phospho-YB1 (Ser102) 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 the absorbance for the developed color is proportional to the quantity of YB1 phosphorylated at Ser102.Antibodies in kit are custom formulations specific to kit.

Specificity / Sensitivity

PathScan® Phospho-YB1 (Ser102) Sandwich ELISA Kit #7249 detects endogenous levels of YB1 protein when phosphorylated at Ser102 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 Jurkat cells, treated with LY294002 (as control) or calyculin A/pervanadate, and the absorbance at 450 nm using the PathScan® Phospho-YB1 (Ser102) Sandwich ELISA Kit is shown.

ELISA - Western correlation

ELISA - Western correlation

Figure 1. Treatment of Jurkat cells with calyculin A and pervanadate stimulates phosphorylation of YB1 at Ser102, detected by the PathScan® Phospho-YB1 (Ser102) Sandwich ELISA Kit #7249, but does not affect the levels of total YB1 detected by western. Jurkat cells were treated with 50 μM LY294002 (as control), or 100 nM calyculin A and 1 mM pervanadate for 30 minutes and lysed. The absorbance readings at 450 nm are shown in the upper figure, while the corresponding western blots using YB1 (D299) Antibody #4202 (left panel) or Phospho-YB1 (Ser102) (C34A2) Rabbit mAb #2900 (right panel) are shown in the lower figure.

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).

  1. Matsumoto, K. and Wolffe, A.P. (1998) Trends Cell Biol. 8, 318-23.
  2. Jurchott, K. et al. (2003) J. Biol. Chem. 278, 27988-96.
  3. Mertens, P.R. et al. (1997) J. Biol. Chem. 272, 22905-12.
  4. Uchiumi, T. et al. (1993) Cell Growth Differ. 4, 147-57.
  5. Lasham, A. et al. (2000) Gene 252, 1-13.
  6. Lasham, A. et al. (2003) J. Biol. Chem. 278, 35516-23.
  7. Homer, C. et al. (2005) Oncogene 24, 8314-25.
  8. Raffetseder, U. et al. (2003) J. Biol. Chem. 278, 18241-8.
  9. Chen, C.Y. et al. (2000) Genes Dev. 14, 1236-48.
  10. Sutherland, B.W. et al. (2005) Oncogene 24, 4281-92.

Application References

Have you published research involving the use of our products? If so we'd love to hear about it. Please let us know!

Protocols

Companion Products

For Research Use Only. Not For Use In Diagnostic Procedures.

Products