Protein kinase assay using tritiated peptide substrates and ferric adsorbent paper for phosphopeptide binding.
The recently described synthesis of ferric adsorbent paper has made possible the modification of protein kinase assays. The adsorbent contains ferric chelate groups, which are responsible for the binding of phosphopeptide via phosphate group. The selective adsorption of phosphopeptide contra nonphosphorylated peptide allows the use of tritium-labeled peptides and unlabeled ATP as substrates. The binding of the reaction product to the adsorbent was complete and was not affected by the amino acid sequence of the peptide. The conditions required for the separation of the produced phosphopeptide from the initial peptide have been worked out as well. The firmly bound phosphopeptide should be released from the ferric adsorbent paper prior to liquid scintillation counting. Using 0.1 m NH4HCO3 solution (pH 8.0), the elution of phosphopeptides was almost complete. The modified protein kinase assay proposed herein is rapid and allows handling of multiple samples simultaneously. In addition, the ferric paper method avoids the use of 32P-isotope, replacing it with 3H which has lower radiation energy and a much longer half-life.
Toomik
R
R
Department of Medical and Physiological Chemistry, Uppsala University, Sweden.
Ekman
P
P
Eller
M
M
Järv
J
J
Zaitsev
D
D
Myasoedov
N
N
Ragnarsson
U
U
Engström
L
L
D000327:Adsorption; D000595:Amino Acid Sequence; D000818:Animals; D002614:Chelating Agents; D002854:Chromatography, Paper; D005290:Ferric Compounds; D008969:Molecular Sequence Data; D010748:Phosphopeptides / Q000737:chemistry*; D010766:Phosphorylation; D011485:Protein Binding; D011494:Protein Kinases / Q000737:chemistry* / Q000302:isolation & purification; D051381:Rats; D012680:Sensitivity and Specificity; D013552:Swine; D014316:Tritium
D002614:Chelating Agents; D005290:Ferric Compounds; D010748:Phosphopeptides; D014316:Tritium; D011494:Protein Kinases