Cell Signaling Technology

Product Pathways - PathScan ELISA

PathScan® Phospho-MEK1 (Ser217/221) Sandwich ELISA Kit #7175

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Kit Includes Volume Solution Color
MEK1 (61B12) mAb Coated Microwells
P-MEK1/2(Ser217/221) Detection Ab 11 ml
Anti-rabbit IgG, HRP-linked Antibody 11 ml
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 M

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

Description

CST's PathScan® Phospho-MEK1 (Ser217/221) Sandwich ELISA Kit is a solid phase sandwich enzyme-linked immunosorbent assay (ELISA) that detects endogenous levels of Phospho-MEK1 (Ser217/221) protein. MEK1 Mouse mAb has been coated onto the microwells. After incubation with cell lysates, total MEK1 protein (phospho- and nonphospho-) is captured by the coated antibody. Following extensive washing, a Phospho-MEK1/2 Antibody is added to detect the captured phospho-MEK1 protein. HRP-linked, anti-rabbit antibody #7074 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 phospho-MEK1 protein.Antibodies in kit are custom formulations specific to kit.

Specificity / Sensitivity

CST's PathScan® Phospho-MEK1 (Ser217/221) Sandwich ELISA Kit detects endogenous levels of phospho-MEK1 protein. A significant induction of MEK1 phosphorylation in PMA-treated NIH/3T3 cells can be detected using this kit, #7175. However, the level of total MEK1 protein detected by PathScan® Total MEK1 Sandwich ELISA Kit #7165 remains unchanged (Figure 1). This kit can also be used to detect phosphorylated MEK1 protein in human 293 cells. 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 PMA stimulates phosphorylation of MEK1 at Ser217/221, detected by PathScan® Phospho-MEK1 (Ser217/221) Sandwich ELISA kit #7175, but does not affect the level of total MEK1 protein detected by PathScan® Total MEK1 Sandwich ELISA kit #7165. Lambda protein phosphatase (LPP) treatment of control cell lysates (37ºC for 90 minutes) abolished the basal phosphorylation of MEK1 in control lysates shown in both Sandwich ELISA and Western analysis. The OD450 readings are shown in the top figure, while the corresponding Western blot, using Phospho-MEK1/2 (Ser217/221) Antibody #9121 (right panel) or MEK1 Mouse mAb #2352 (left panel), is shown in the bottom figure.

Sensitivity

Sensitivity

Figure 2: Linear relationship between protein concentration of lysates from untreated and PMA-treated NIH/3T3 cells and kit assay optical density readings. Cells (80% confluence) were treated with PMA (120 ng/ml) and lysed after incubation at 37ºC for 30 minutes. Lambda protein phosphatase (LPP) treatment of control cell lysates was performed at 37ºC for 90 minutes.

Background

MEK1 and MEK2, also called MAPK or Erk kinases, are dual-specificity protein kinases that function in a mitogen activated protein kinase cascade controlling cell growth and differentiation (1-3). Activation of MEK1 and MEK2 occurs through phosphorylation of two serine residues at positions 217 and 221, located in the activation loop of subdomain VIII, by Raf-like molecules. MEK1/2 is activated by a wide variety of growth factors and cytokines and also by membrane depolarization and calcium influx (1-4). Constitutively active forms of MEK1/2 are sufficient for the transformation of NIH/3T3 cells or the differentiation of PC-12 cells (4). MEK activates p44 and p42 MAP kinase by phosphorylating both threonine and tyrosine residues at sites located within the activation loop of kinase subdomain VIII.

  1. Crews, C.M. et al. (1992) Science 258, 478-480.
  2. Alessi, D.R. et al. (1994) EMBO J. 13, 1610-1619.
  3. Rosen, L.B. et al. (1994) Neuron 12, 1207-1221.
  4. Cowley, S. et al. (1994) Cell 77, 841-852.

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