FAQ: Enzyme Profiling - Substrate Sets (Peptide Collections)

JPT Peptide Technologies provides a detailed protocol together with every Enzyme Substrate Set including plate layout and sequence list in Excel-format.
The protocols can also be downloaded from our website.

The peptides used in the Kinase Substrate Sets are N-terminally biotinylated and C-terminally amidated.
The peptides used in the Phosphatase Substrate Sets are N-terminally free and C-terminally amidated.
The peptides used in the Protease Substrate Sets carry Fluorophore and Quencher on N- and C-terminal ends respectively.

The enzyme concentration is mainly depending on the specific activity of your enzyme.
If you already have experimental data on you enzyme please use these as orientation.
If there is no information on enzyme activity available, we recommend to start with a high concentration and adjust later if necessary.

Peptide quantities are optimized for a single use.

All kinase substrates are available in larger quantities and can be purchased off-the-shelf.
For phosphatase and protease substrates customized synthesis is necessary and can be ordered fast and economically.
Please contact our customer support at peptide@jpt.com.

Generally, most peptides are stable over years when stored at -20°C in freeze dried state.
However, peptides containing amino acids such as Met, Cys or N-terminal Gln tend to oxidation or pyroglutamate formation.
Since kinetics of such deterioration depends on primary peptide structure, stability can hardly be predicted.
Therefore, we recommend usage of peptide sets within 6 months.
Once thawed and dissolved in buffer solution, JPT recommends performing the assay without further delay to avoid degradation.

The plates are shipped freeze dried at room temperature.
However, for long-term storage, JPT Peptide Technologies recommends storage at -20°C.

For Kinase Substrate Sets, the kinase in question, radioisotopic labeled ATP, as well as assay buffer are needed.
If fluorescence read-out should be performed, a suitable fluorescently labeled antibody or phosphospecific stain is needed.

For Phosphatase Substrate Sets, the phosphatase in question and the appropriate assay buffer are needed.
As read-out reagent JPT peptide Technologies recommends "Biomol green". For more details, please refer to the protocol.

For Protease Substrate Set, the protease in question and the appropriate assay buffer are needed.

For radioisotobic read-out of the kinase substrate set, a Streptavidin membrane (Promega, SAM-membrane) is needed, as well as a system for detecting incorporated radioisotopic labeled Phosphate (Phospho-Imager).
For the Phosphatase Substrate Sets, a microtiterplate-reader capable of detecting absorbance at 620-660nm is needed.
For the Protease Substrate Sets, a microtiterplate-reader capable of detecting fluorescence (Ex: 350nm, Em: 490nm). Preferred read-out procedure is from top but microtiterplates with clear bottom are also available.