Figure 2. The relationship between the protein concentration of lysates from HeLa cells, untreated or treated with TPA #4174, and the absorbance at 450 nm using the PathScan® Phospho-RSK1 (Ser380) Sandwich ELISA Kit is shown.
Figure 1. Treatment of HeLa cells with TPA stimulates phosphorylation of RSK1 at Ser380, detected by the PathScan® Phospho-RSK1 (Ser380) Sandwich ELISA Kit #7965. HeLa cells (80-90% confluent) were treated with 200 nM TPA #4174 for 30 minutes at 37ºC. The absorbance readings at 450 nm are shown in the top figure, while the corresponding western blots using RSK1 Antibody #9333 (left panel) or Phospho-p90RSK (Ser380) (9D9) Rabbit mAb #9335 (right panel) are shown in the bottom figure.
|Product Includes||Volume||Solution Color|
|Phospho-Rsk (Ser380) Rabbit mAb Coated Microwells||96 tests|
|RSK Mouse Detection mAb||1 ea||Green (Lyophilized)|
|Anti-mouse IgG, HRP-linked Antibody (ELISA Formulated)||1 ea||Red (Lyophilized)|
|Detection Antibody Diluent||11 ml||Green|
|HRP Diluent||11 ml||Red|
|TMB Substrate 7004||11 ml|
|STOP Solution 7002||11 ml|
|Sealing Tape||2 ea|
|ELISA Wash Buffer (20X) 9801||25 ml|
|ELISA Sample Diluent||25 ml||Blue|
|PathScan® Sandwich ELISA Lysis Buffer (1X) 7018||30 ml|
PathScan® Phospho-RSK1 (Ser380) Sandwich ELISA Kit from Cell Signaling Technology is a solid phase sandwich enzyme-linked immunosorbent assay (ELISA) that detects endogenous levels of RSK1 when phosphorylated at Ser380. A Phospho-RSK(Ser380) rabbit antibody has been coated onto the microwells. After incubation with cell lysates, phospho-RSK1 proteins is captured by the coated antibody. Following extensive washing, a RSK1 mouse detection antibody is added to detect the captured RSK1 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 RSK1 phosphorylated at Ser380.
Antibodies in kit are custom formulations specific to kit.
NOTE: Prepare solutions with purified water.
*NOTE: Some PathScan® ELISA Kits may include HRP-Linked Streptavidin in place of HRP-Linked Antibody.
NOTE: Initial color of positive reaction is blue, which changes to yellow upon addition of STOP Solution.
posted November 2013
revised January 2016
Protocol Id: 205
PathScan® Phospho-RSK1 (Ser380) Sandwich ELISA Kit #7965 detects endogenous levels of RSK1 protein when phosphorylated at Ser380. As shown in Figure 1, a significant induction of RSK1 phosphorylation at Ser380 can be detected in HeLa cells following treatment with TPA using the Phospho-RSK1 (Ser380) Sandwich ELISA Kit #7965. The levels of total RSK1 (phospho and nonphospho) remain unchanged, as shown by western analysis and by PathScan®Total RSK1 Sandwich ELISA Kit #7966 (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:
The 90 kDa ribosomal S6 kinases (RSK1-4) are a family of widely expressed Ser/Thr kinases characterized by two nonidentical, functional kinase domains (1) and a carboxy-terminal docking site for extracellular signal-regulated kinases (ERKs) (2). Several sites both within and outside of the RSK kinase domain, including Ser380, Thr359, Ser363, and Thr573, are important for kinase activation (3). RSK1-3 are activated via coordinated phosphorylation by MAPKs, autophosphorylation, and phosphoinositide-3-OH kinase (PI3K) in response to many growth factors, polypeptide hormones, and neurotransmitters (3).
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Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
PathScan is a trademark of Cell Signaling Technology, Inc.
U.S. Patent No. 7,429,487, foreign equivalents, and child patents deriving therefrom.