Western blot analysis of Elk-1 fusion protein expressed from E. coli with or without phosphorylation by purified Erk2 enzyme, using Phospho-Elk-1 (Ser383) Antibody #9186 (upper) or control Elk-1 Antibody #9182 (lower).
Boil for 3 minutes prior to use. Load 15 µl of phosphorylated and nonphosphorylated Elk1 Control Proteins.
Supplied in SDS Sample Buffer: 62.5 mM Tris-HCl (pH 6.8 at 25°C), 2% w/v SDS, 10% glycerol, 50 mM DTT, 0.01% w/v phenol red or bromophenol blue. Store at –20°C or at –80°C for long term storage.
Nonphosphorylated Elk-1 Control Proteins: Bacterially expressed Elk1 fusion protein serves as a negative control. Supplied in SDS Sample Buffer.
Phosphorylated Elk-1 Control Proteins: Bacterially expressed Elk1 fusion protein phosphorylated by the Erk2 enzyme serves as a positive control. Supplied in SDS Sample Buffer.
The transcription factor Elk-1 is a component of the ternary complex that binds the serum response element (SRE) and mediates gene activity in response to serum and growth factors (1-3). Elk-1 is phosphorylated by MAP kinase pathways at a cluster of S/T motifs at its carboxy terminus; phosphorylation at these sites, particularly Ser383, is critical for transcriptional activation by Elk-1. Elk-1 appears to be a direct target of activated MAP kinase: (a) biochemical studies indicate that Elk-1 is a good substrate for MAP kinase; (b) the kinetics of Elk-1 phosphorylation and activation correlate with MAP kinase activity; (c) interfering mutants of MAP kinase block Elk-1 activation in vivo. Other studies have shown that Elk-1 (Ser383) is also a target of the stress-activated kinase SAPK/JNK (4,5).
Western Blots: For controls, we recommend using 15 µl of phosphorylated and nonphosphorylated Elk1 control extracts. Boil sample prior to loading.
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