Applications
- Identification and optimization of kinase-, phosphatase-,
acetyltransferase- and histone deacetylase-substrates via
standard screening systems (AlphaScreen, FlashPlates, SPA-Beads,
and many more).
- Mapping of protein/protein interaction sites
(ELISA-like
assays, precipitation of interacting proteins).
- Production of peptide microarrays.
- Loading of columns for affinity chromatography.
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Example: Human c-Jun derived BioTides and
protein tyrosine kinase c-Abl
Used reagents and materials:
- 384 well microtiter plate containing 160 different BioTides
derived from human c-Jun protein
- Abl-kinase (New England BioLabs, www.neb.com, item#
P6050L)
- Kinase buffer 50 mM Tris-HCl, 10 µM MgCl2 1 mM EGTA,
2 mM DTT, and 0.01% Brij 35 (pH 7.5, 25°C)
- SAM-Biotin capture membrane (Promega,
# V7861)
- 384 pin replicator (Genetix,
384-pin QReps-PP, Product Code: X5050, printing volume
approx: 0.2 µl)
Wells of a microtiter plate each containing 1 nanomole BioTide
were incubated with 2 Units c-Abl in the presence of 400 µM
ATP using 20 µl kinase buffer per well for 3h at 30°C. The
resulting final BioTide concentration was 50 µM. Subsequent
to incubation aliquots of assay solution were printed directly
on SAM-Biotin capture membrane using a 384 pin replicator followed
by washings (two times) with TBS-buffer (50 mM Tris-HCl, 137
mM NaCl, 2,7 mM KCl (pH 8, 25°C). The SAM-membrane was blocked
for 1h with blocking reagent from Roche Diagnostics (dilution
1:10, # 1 096 176)
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