Kinase Substrate Optimization

Peptide microarrays are a valuable tool for kinase substrate optimization. The peptide array covers a single substitution scan of the phosphorylation site and is incubated with the active tyrosine kinase or serine/threonine kinase in the presence of ATP. Phosphorylated tyrosines, serines or threonines will be selectively stained with a fluorescently labeled tag. Peptide variants that are less or stronger phosphorylated than the original substrate can be identified.


 

Phosphorylation of two peptide substrate families by c-Src. The sequences of the strongest peptide variants were listed as top hits. Peptide substrates were further classified according to their spot intensities in background (-), weak (1,000-3,000 FU; +), medium (3,000 – 10,000 FU; ++), strong (10,000 – 20,000 FU; +++) and very strong (> 20,000 FU; ++++) signals; amino acid exchanges compared to the starting substrate were shown in green.



Specifications

  • Array content: Successive replacement of the amino acid positions -2, -1, +1, +2 next to the phosphorylation site of the starting peptide by all 19 other L-amino acids
  • Control peptides: HA peptides in the array frame and polyEY and Poly EAY in the center of the array

 

 

Input Data & Sample Material

  • Peptide sequence of kinase substrate

  • 100 µg purified and active kinase



Analysis

Kinase substrate optimization is provided along with our comprehensive PEPperMAP® Service to ensure optimal results.

 

 

 

Further Information

 

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