Cell Signaling Technology

Product Pathways - PathScan ELISA

PathScan® Total RSK1 Sandwich ELISA Kit #7966

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

Kit Includes Volume Solution Color
Rsk1 Ab Coated Microwells 96 assays
Rsk1 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 Wash Buffer (20X) 25 ml Colorless
ELISA Sample Diluent 25 ml Blue
PathScan® Sandwich ELISA Lysis Buffer (1X) #7018 30 ml Colorless

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

PathScan® Total RSK1 Sandwich ELISA Kit from Cell Signaling Technology is a solid phase sandwich enzyme-linked immunosorbent assay (ELISA) that detects endogenous levels of total RSK1 protein. A RSK1 Rabbit mAb has been coated onto the microwells. After incubation with cell lysates, both phospho- and nonphospho-RSK1 proteins are captured by the coated antibody. Following extensive washing, a RSK1 Mouse Antibody is added to detect both the captured phospho- and nonphospho-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 optical density for this developed color is proportional to the quantity of total RSK1 protein.Antibodies in kit are custom formulations specific to kit.

Specificity / Sensitivity

CST's PathScan® Total RSK1 Sandwich ELISA Kit #7966 detects endogenous levels of total RSK1 protein. 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.

ELISA

ELISA

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® Total RSK1 Sandwich ELISA Kit is shown.

ELISA - Western correlation

ELISA - Western correlation

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, but does not affect the levels of total RSK1 detected by PathScan® Total RSK1 Sandwich ELISA Kit #7966. HeLa cells (80-90% confluent) were treated with 200 nM TPA #4174 for 30 minutes. 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.

Background

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).

  1. Fisher, T.L. and Blenis, J. (1996) Mol Cell Biol 16, 1212-9.
  2. Smith, J.A. et al. (1999) J Biol Chem 274, 2893-8.
  3. Dalby, K.N. et al. (1998) J Biol Chem 273, 1496-505.

Application References

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Protocols

Companion Products

Rabbit monoclonal antibody is produced under license (granting certain rights including those under U. S. Patents No. 5,675,063 and7,429,487) from Epitomics, Inc.

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

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