Cell Signaling Technology

Product Pathways - PathScan ELISA

PathScan® Phospho-GSK-3β (Ser9) Sandwich ELISA Kit #7311

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Kit Includes Volume Solution Color
GSK-3ß Mouse mAb coated microwells 96 tests
Phospho-GSK-3ß (Ser9) Rabbit Detection mAb 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 Wash Buffer (20X) 25 ml Colorless
ELISA Sample Diluent 25 ml Blue
Cell Lysis Buffer (10X) #9803 15 ml

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 M R Mk

Reactivity Key:  H=Human  M=Mouse  R=Rat  Mk=Monkey
Species enclosed in parentheses are predicted to react based on 100% sequence homology.

Description

The PathScan® Phospho-GSK-3β (Ser9) Sandwich ELISA Kit is a solid phase sandwich enzyme-linked immunosorbent assay (ELISA) that detects endogenous levels of GSK-3β protein phosphorylated at Ser9. A GSK-3β mouse mAb has been coated onto the microwells. After incubation with cell lysates, GSK-3β (phospho and nonphospho) protein is captured by the coated antibody. Following extensive washing, a phospho-GSK-3β (Ser9) rabbit mAb is added to detect the captured phospho-GSK-3β 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 the absorbance for this developed color is proportional to the quantity of GSK-3β phosphorylated at Ser9.Antibodies in kit are custom formulations specific to kit.

Specificity / Sensitivity

PathScan® Phospho-GSK-3β (Ser9) Sandwich ELISA Kit #7311 detects endogenous levels of human GSK-3β protein when phosphorylated at Ser9 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.

ELISA - Western correlation

ELISA - Western correlation

Figure 1. Treatment of NIH/3T3 cells with hPDGF-BB #8912 stimulates phosphorylation of GSK-3β at Ser9, as detected by PathScan® Phospho-GSK-3β (Ser9) Sandwich ELISA Kit #7311, but does not affect levels of total GSK-3β protein detected by PathScan® Total GSK-3β Sandwich ELISA Kit #7265. The absorbance readings at 450 nm are shown in the upper figure, while the corresponding western blots using GSK-3β (27C10) Rabbit mAb #9315 (left panel) or Phospho-GSK-3β (Ser9) (D85E12) XP® Rabbit mAb #5558 (right panel) is shown in the lower figure.

Sensitivity

Sensitivity

Figure 2. The relationship between protein concentration of lysates from untreated and PDGF-treated NIH/3T3 cells and the absorbance at 450 nm is shown. After starvation, NIH/3T3 cells (85% confluence) were treated with hPDGF-BB #8912 (50 ng/ml) for 10-15 minutes at 37ºC and then lysed.

Background

Glycogen synthase kinase-3 (GSK-3) was initially identified as an enzyme that regulates glycogen synthesis in response to insulin (1). GSK-3 is a ubiquitously expressed serine/threonine protein kinase that phosphorylates and inactivates glycogen synthase. GSK-3 is a critical downstream element of the PI3K/Akt cell survival pathway whose activity can be inhibited by Akt-mediated phosphorylation at Ser21 of GSK-3α and Ser9 of GSK-3β (2,3). GSK-3 has been implicated in the regulation of cell fate in Dictyostelium and is a component of the Wnt signaling pathway required for Drosophila, Xenopus, and mammalian development (4). GSK-3 has been shown to regulate cyclin D1 proteolysis and subcellular localization (5).

  1. Welsh, G.I. et al. (1996) Trends Cell. Biol. 6, 274-279.
  2. Srivastava, A.K. and Pandey, S.K. (1998) Mol. Cell. Biochem. 182, 135-141.
  3. Cross, D.A. et al. (1995) Nature 378, 785-789.
  4. Nusse, R. (1997) Cell 89, 321-323.
  5. Diehl, J.A. et al. (1998) Genes Dev. 12, 3499-3511.

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