View - ResearchGate

204 Urh et al.
The following protocol was used with HeLa cells cultured in 10-cm
Petri dish transfected with pFC8A(HT)-p65 (encoding human p65-HaloTag
fusion protein). This protocol used a lipid-based transfection reagent and was
performed according to the manufacturer’s instructions.
3.3.1. Day 1: Plating Cells
HeLa cells (1.5–2.5 × 10 6 cells) were plated in 10-cm plastic Petri dish and
grown overnight in Dulbecco’s Modified Eagle’s Medium + 10% fetal bovine
serum in atmosphere of 5% C0 2 at 37°C to 70–80% density.
3.3.2. Day 2: Transfecting Cells
Transfect cells following the manufacturer’s instructions for the transfection
reagent that you are using. In our case, cells were transfected using Lipofectamine
2000 according to manufacturer’s protocol using 1–2 μg DNA and
50 μl of Lipofectamine 2000 per dish.
3.3.3. Day 3: Capturing and Analysis of the Protein Complexes
3.3.3.1. Phase 1: Preparation of Cytosolic Fraction
1. Twenty-four-hour post-transfection, aspirate off media and wash cells twice with
5 ml of ice-cold 10 mM N-(2 Hydroxyethyl piperazine-N´-(2-ethanesulfonic acid);
4-(2 Hydroxyethyl) piperazine-1-ethanesulfonic acid (HEPES) buffer, pH 7.5.
2. Resuspend cells in 1 ml of the HEPES buffer containing protease inhibitors and
collect by scraping.
3. Lyse cells using mechanical disruption (e.g., use glass homogenizer 2 ml size;
25–30 strokes on ice or through a 27-guage needle) followed by sonication on ice.
4. Centrifuge at 10,000 × g for 7 min at 4°C.
5. Carefully remove supernatant and use immediately or store at –70°C for up to a
month.
3.3.3.2. Phase 2: Resin Equilibration
Mix resin by inverting to obtain uniform suspension.
1. Dispense 100 μl of HaloLink resin into 1.5-ml Eppendorf tube and spin in
microcentrifuge for 1 min at 800 × g.
2. Carefully remove and discard the supernatant leaving resin at the bottom of the
tube.
3. Add 400 μl of binding buffer, mix thoroughly by inverting the tube.
4. Centrifuge for 2 min at 800 × g.
5. Carefully remove and discard the supernatant leaving resin at the bottom of the tube.
6. Repeat steps 3–5 two more times for a total of three washes.
7. After last wash, resuspend the resin in 40 μl of binding buffer.