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.
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)
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.
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.
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).
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