This Mouse MEK1 overexpression lysate was created in Baculovirus-Insect cells and intented for use as a Western blot (WB) positive control. Purification of MEK1 protein (Cat: 50312-M20B) from the overexpression lysate was verified.
A DNA sequence encoding the mouse MAP2K1 (P31938) (Met 1-Ile 393) was fused with the N-terminal polyhistidine-tagged GST tag at the N-terminus.
The recombinant mouse MAP2K1/GST chimera consists of 630 amino acids and has a calculated molecular mass of 71.3 kDa. It migrates as an approximately 65 kDa band in SDS-PAGE under reducing conditions.
Cell lysate was prepared by homogenization of the over-expressed cells in ice-cold modified RIPA Lysis Buffer with cocktail of protease inhibitors (Sigma). Cell debris was removed by centrifugation. Protein concentration was determined by Bradford assay (Bio-Rad protein assay, Microplate Standard assay). The cell lysate was boiled for 5 min in 1 x SDS loading buffer (50 mM Tris-HCl pH 6.8, 12.5% glycerol, 1% sodium dodecylsulfate, 0.01% bromophenol blue) containing 5% b-mercaptoethanol, and lyophilized.
MEK1, also known as MAP2K1 and MKK1, is a member of the dual-specificity protein kinase family, which acts as a mitogen-activated protein (MAP) kinase kinase. MAP kinases, also known as extracellular signal-regulated kinases (ERKs), act as an integration point for multiple biochemical signals. MEK1 is widely expressed, with extremely low levels in the brain. It lies upstream of MAP kinases and stimulates the enzymatic activity of MAP kinases upon a wide variety of extra- and intracellular signals. As an essential component of the MAP kinase signal transduction pathway, MEK1 is involved in many cellular processes such as proliferation, differentiation, transcription regulation, and development. Binding extracellular ligands such as growth factors, cytokines, and hormones to their cell-surface receptors activates RAS and this initiates RAF1 activation. RAF1 then further activates the dual-specificity protein kinases MAP2K1 and MEK2. MEK1 has been shown to export PPARG from the nucleus. The MAPK cascade is also involved in the regulation of endosomal dynamics, including lysosome processing and endosome cycling through the perinuclear recycling compartment (PNRC), as well as in the fragmentation of the Golgi apparatus during mitosis. MKK1 catalyzes the concomitant phosphorylation of a threonine and a tyrosine residue in a Thr-Glu-Tyr sequence located in MAP kinases. Defects in MEK1 can cause Cardiofaciocutaneous Syndrome.