Kinase assay reagents
• kinase assay buffer: Cell Signaling cat.# 9802
• ATP solution: Cell Signaling cat.# 9804
• Kinase-Glo revealing reagent: Promega; cat.# V3772
• 384 well-plates: solid white flat bottom plates, Corning cat.# 3570
• micro-centrifuge tubes: Costar; cat.# 3621
• clear 96-well PCR-plates: VWR; cat.# 83007-374
Peptides (which serve as surrogate sensors of kinases’ phospho-catalytic enzymatic activity)
• Mass-scale chemical synthesis (either in-house or outsourced) of 11-mer amino acid sequences at >95% purity.
• Biological peptides, defined as an amino acid sequence corresponding to a known phosphorylatable protein region for a biological peptide27, can be available from curated kinase – substrate – phospho-target site relationship databases, especially PhosphoAtlas27 (http://cancer.ucsf.edu/phosphoatlas; DOI: 10.1158/0008-5472.CAN-15-2325-T), or other resources (e.g. Phospho Site Plus26).
• Generic positive control peptides, defined as commercially available amino acid sequence advertised as a kinase probe/sensor for a CON+ peptide, can be available from any manufacturer (e.g. SignalChem: Abltide, cat.# A02-58; or Poly (4:1 Glu, Tyr) peptide, cat# P61-58).
• Reference peptides, defined as (i) modified biological or generic positive control peptides either mutating Tyrosine (Y) / Serine (S) / Threonine (T) sites replaced with a Glycine (G), and/or pre-phosphorylated Y / S / T sites replaced with pY / pS / pT), and (ii) random peptide sequences (e.g. amino acid-repeated sequence).
Recombinant kinases and kinase inhibitors
• If needed, recombinant kinases and kinase inhibitors can either be internally produced/purified by a laboratory or a facility, or purchased from providers (e.g. SignalChem, Selleck Chemicals, Invitrogen, Tocris, Sigma-Aldrich, EMD-CalBiochem, etc).
Reagents to generate protein extracts from biological samples (cultured cells or tissues)
• Cell Lysis Buffer (i.e. non-denaturing lysis buffer): Cell Signaling, cat.# 9803
• Halt Protease & Phosphatase: ThermoScientific, cat.# 1861281