Cell Signaling Technology

Product Pathways - PathScan ELISA

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

Kit Includes Volume Solution Color
MEK1 (61B12) Mouse mAb Coated Microwells
Phospho-MEK1/2 Detection Antibody
Anti-rabbit IgG HRP-Linked Antibody
TMB Substrate 11 milliliters Colorless
STOP Solution 11 milliliters Colorless
Sealing Tape 2 sheets
20X Wash Buffer 25 milliliters Colorless
Sample Diluent 25 milliliters Blue
Cell Lysis Buffer (10X) # 9803 15 milliliters 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 (10X), which is stored at –20°C (packaged separately).

Species Cross-Reactivity

H M

Reactivity Key:  H=Human  M=Mouse

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 (61B12) Mouse mAb #2352* 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 #9121* 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 this kit are custom formulations specific to the the 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.

Western Blotting

Western Blotting

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 (37°C 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.

Sandwich ELISA

Sandwich ELISA

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 37°C for 30 minutes. Lambda protein phosphatase (LPP) treatment of control cell lysates was performed at 37°C 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 (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 PC12 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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