Cell Signaling Technology

Product Pathways - PathScan ELISA

PathScan® Phospho-Survivin (Thr34) Sandwich ELISA Kit #7193

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Kit Includes Volume Solution Color
Phospho-Survivin (Thr34) Rabbit Antibody Coated Microwells 96 tests
Survivin Mouse Detection Antibody 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 Wash Buffer (20X) 25 ml Colorless
ELISA Sample Diluent 25 ml Blue
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-Survivin (Thr34) Sandwich ELISA Kit is a solid phase sandwich enzyme-linked immunosorbent assay (ELISA) that detects endogenous levels of survivin protein phosphorylated at Thr34. A phospho-survivin (Thr34) rabbit antibody has been coated onto the microwells. After incubation with cell lysates, phospho-survivin protein is captured by the coated antibody. Following extensive washing, a survivin mouse antibody is added to detect the captured phospho-survivin 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 this developed color is proportional to the quantity of survivin phosphorylated at Thr34.Antibodies in kit are custom formulations specific to kit.

Specificity / Sensitivity

CST's PathScan® Phospho-Survivin (Thr34) Sandwich ELISA Kit #7193 detects endogenous levels of survivin protein phosphorylated at Thr34 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 - Western correlation

ELISA - Western correlation

Figure 1: HeLa cells synchronized with nocodazole accumulate phosphorylation of survivin at Thr34, as detected by PathScan® Phospho-Survivin (Thr34) Sandwich ELISA Kit #7193. The accumulated total survivin protein can be detected by PathScan® Total Survivin Sandwich ELISA Kit #7169. The absorbance readings at 450 nm are shown in the top figure, while the corresponding western blots using Survivin Mouse mAb #2802 (left panel) or Phospho-Survivin (Thr34) Rabbit Antibody (right panel) are shown in the bottom figure.

Sensitivity

Sensitivity

Figure 2: The relationship between protein concentration of lysates from untreated and nocodazole-treated HeLa cells and the absorbance at 450 nm is shown. After starvation, HeLa cells (85% confluence) were synchronized with nocodazole (50 ng/ml) for 20 hr at 37ºC and then lysed. Added 1X Phosphatase Inhibitor Cocktail (100X) #5870 to lysis buffer in addition to the 1mM phenyl-methylsulfonyl fluoride (PMSF)

Background

Survivin is a 16 kDa anti-apoptotic protein highly expressed during fetal development and cancer cell malignancy (1). Survivin binds and inhibits caspase-3, controlling the checkpoint in the G2/M-phase of the cell cycle by inhibiting apoptosis and promoting cell division (2,3). This regulatory process requires the phosphorylation of survivin at Thr34 by p34 cdc2 kinase (4). Gene targeting using a Thr34 phosphorylation-defective survivin mutant, as well as antisense survivin, have been shown to inhibit tumor growth (5,6).

  1. Reed, J.C. and Reed, S.I. (1999) Nature Cell Biol. 1, 199-200.
  2. Li, F. et al. (1998) Nature 396, 580-584.
  3. Li, F. et al. (1999) Nat. Cell Biol. 1, 461-466.
  4. O'Connor, D.S. et al. (2000) Proc. Natl. Acad. Sci. USA 97, 13103-13107.
  5. Olie, R.A. et al. (2000) Cancer Res. 60, 2805-2809.
  6. Grossman, D. et al. (2001) Proc. Natl. Acad. Sci. USA 98, 635-640.

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