Figure 2. Elk-1 fusion protein was used as substrate to measure Erk1 kinases activity in a radiometric assay using the following reaction conditions: 25 mM Tris-HCl (pH7.5), 10 mM MgCl2, 5 mM b-glycerophosphate, 0.1 mM Na3VO4, 2 mM DTT, 50 μM ATP, Substrate: Elk-1 fusion protein 400 ng/μL, and variable amounts of Erk1 kinases.
|MW (kDa)||41, 45|
Elk-1 Fusion Protein serves as a useful substrate for p42/44 MAP kinases (1,6). It is expressed as a recombinant protein fusion containing Elk-1 residues 307–428.
Apparent Molecular Weight: 41 kDa
Elk 1 Fusion Protein at a concentration of 2 µg/20 µ| reaction was phosphorylated using p42 MAP kinase (Erk1) in an in vitro kinase assay with 1X Kinase Buffer #9802 and 200 µM ATP #9804. After a 30-minute assay at 30°, phosphorylation was detected by Western blot with Phospho-Elk-1 (Ser383) Antibody #9181.
Elk 1 Fusion Protein, at a concentration of 2 µg/20 µl reaction, can be phosphorylated using 50 units of p42 MAP kinase (Erk2) in an in vitro kinase assay with 1X Kinase Buffer #9802 and 200 µM ATP #9804. After a 30-minute assay at 30ºC, phosphorylation can be detected by Western blot with Phospho-Elk-1 (Ser383) Antibody #9181.
Store at -20°C.
The purified fusion protein was identified by SDS-PAGE and Western blot analysis, using Elk-1 Antibody #9182, to be greater than 95% pure Elk-1 .
Cloned from a human cDNA library (7) and overexpressed in E. coli.d in E. coli.
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).
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