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.
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.
|Product Includes||Volume||Solution Color|
|MEK1/2 Rabbit mAb Coated Microwells||96 tests|
|MEK1 Mouse Detection mAb||1 ea||Green (Lyophilized)|
|Anti-mouse IgG, HRP-linked Antibody (ELISA Formulated)||1 ea||Red (Lyophilized)|
|Detection Antibody Diluent 2||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|
|Cell Lysis Buffer (10X) 9803||15 ml|
CST's PathScan® Total MEK1 Sandwich ELISA Kit is a solid phase sandwich enzyme-linked immunosorbent assay (ELISA) that detects endogenous levels of total MEK1 protein. A 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, MEK1/2 Antibody is added to detect the captured 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 total MEK1 protein.
Antibodies in kit are custom formulations specific to kit.
NOTE: Refer to product-specific datasheets or product webpage for assay incubation temperature.
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.
1X Cell Lysis Buffer: 10X Cell Lysis Buffer (#9803): To prepare 10 ml of 1X Cell Lysis Buffer, add 1 ml of 10X Cell Lysis Buffer to 9 ml of dH2O, mix. Buffer can be stored at 4°C for short-term use (1–2 weeks).
Recommended: Add 1 mM phenylmethylsulfonyl fluoride (PMSF) (#8553) immediately before use.
NOTE: Refer to product-specific datasheet or webpage for lysis buffer recommendation.
Add 100 µl of STOP solution to each well. Shake gently for a few seconds.
NOTE: Initial color of positive reaction is blue, which changes to yellow upon addition of STOP solution.
posted June 2005
revised November 2013
Protocol Id: 21
CST's PathScan® Total MEK1 Sandwich ELISA Kit detects endogenous levels of total MEK1 protein. A significant induction of MEK1 phosphorylation can be detected in PMA-treated NIH/3T3 cells using PathScan® Phospho- MEK1 (Ser217/221) Sandwich ELISA Kit #7175. However, the level of total MEK1 detected by this sandwich ELISA kit #7165 remains unchanged (Figure 1). This kit can also be used to detect total 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.
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.
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PathScan is a trademark of Cell Signaling Technology, Inc.