- Purified recombinant target protein. Target protein can be
purified from cell lysate or purchased commercially. Store at
80 C or according to the manufacturer’s instructions, and
thaw on ice prior to use (seeNote 1). - [γ-^32 P] ATP: 250μCi (3000 Ci/mmol) diluted to 10 mCi/ml
and shielded in lead [1](seeNote 2). - Adenosine 5^0 -monophosphate (AMP) solution: 10 mM AMP
in distilled water. Store at 80 C and thaw on ice prior to
use [ 1]. - Magnesium chloride (MgCl 2 ) solution: 250 mM MgCl 2 in
distilled water. Store on ice. - Heating block: must be capable of holding a constant temper-
ature between 37C and 100C. - Centrifuge tubes: must hold at least 50μl total volume.
- Buffer A: 50 mM HEPES, pH 7.4 (seeNotes 3and 4 ).
Protein Separation- SDS-PAGE gels: commercially available.
- Tris-glycine running buffer: 0.025 M Tris, 0.192 M glycine,
0.1% (w/v) SDS prepared in distilled water or obtained
commercially. - 4Laemmli sample buffer: 200 mM Tris–HCl, pH 6.8, 8%
(w/v) SDS, 40% (v/v) glycerol, 4% (v/v)β-mercaptoethanol,
0.05 M EDTA, 0.08% (w/v) bromophenol blue prepared in
distilled water or obtained commercially. - Vertical SDS-PAGE electrophoresis system: commercially
available. - Orbital shaker or rocker: commercially available.
- SDS-PAGE molecular weight markers: commercially available.
- Coomassie R-250 stain: 0.1% (w/v) Coomassie Brilliant Blue
R-250, 5% (v/v) methanol prepared in 10% (v/v) glacial acetic
acid and filtered through Whatman filter paper with a mini-
mum grade of one to remove any aggregated stain. It is also
commercially available. - Destaining solution: 5% (v/v) methanol, 10% (v/v) acetic acid
diluted in distilled water or commercially available.
Film Development- Whatman paper: grade one or equivalent wide enough to cover
the entire gel. - Transparency sheets: commercially available.
- Laboratory tape: commercially available.
- Autoradiograph cassette: commercially available.
Identification and Validation of Novel Targets 101