2.3 Preparation
of Beads
1. IP lysis buffer w/o inhibitors: 50 mM Tris–HCl pH 7.5,
150 mM NaCl, 4 mM MgCl 2 , 0.25% Igepal, 0.25% sodium
desoxycholate, 1% SDS. Add 5 mM DTT before use.
2. Sepharose beads.
3. GFP-Trap
® agarose beads (Chromotek).
4. End-over-end rotator.
5. DNA LoBind
® tubes (Eppendorf).
2.4 Total Extract
Preparation
1. Liquid nitrogen.
2. 0.1 M Phenylmethylsulfonyl fluoride (PMSF) in isopropanol.
3. IP lysis buffer: 50 mM Tris–HCl pH 7.5, 150 mM NaCl, 4 mM
MgCl 2 , 0.25% Igepal, 0.25% sodium desoxycholate, 1% SDS.
Add 5 mM DTT, 100 U RiboLock/mL, 1 mM PMSF, as well
as protease inhibitor tablets, as suggested by the supplier
before use.
4. Steel balls ø 4 mm.
5. 0.45 μM Syringe filter.
6. 5 mL Syringe.
7. Thermomixer (e.g., Eppendorf).
2.5 Immunoprecipitation
1. IP wash buffer: 50 mM Tris–HCl pH 7.5, 500 mM NaCl, 2 M
urea, 4 mM MgCl 2 , 0.5% Igepal, 0.5% sodium desoxycholate,
1% SDS. Add 2 mM DTT and protease inhibitor tablets as
suggested by the supplier before use.
2. PNK wash buffer (pH 7.4): 20 mM Tris–HCl, pH 7.4, 10 mM
MgCl 2 , 0.2% Tween 20.
3. End-over-end rotator.
4. DNA LoBind
® tubes (Eppendorf).
2.6 RNase Digest
1. RNase I (100 U/μL).
2. PNK wash buffer (pH 7.4) (see Subheading 2.5).
3. Turbo DNase (Thermo Fisher Scientific).
2.7 Dephosphorylation
1. 5Â PNK buffer (pH 6.5): 350 mM Tris–HCl, pH 6.5, 50 mM
MgCl 2 , 25 mM DTT (see Note 1).
2. T4 polynucleotide kinase.
3. RiboLock RNase Inhibitor (Thermo Fisher Scientific).
4. PNK wash buffer (pH 7.4) (see Subheading 2.5).
5. High-salt buffer: 50 mM Tris–HCl, pH 7.4, 1 M NaCl, 1 mM
EDTA, 1% Igepal, 0.1% SDS, 0.5% sodium desoxycholate.
6. Thermomixer (e.g., Eppendorf).
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Tino Ko ¨ ster and Dorothee Staiger