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200 Urh et al.
3.2.1.2. Phase 2
Immobilization of HaloTag fusion protein onto HaloLink resin: For
each experimental sample, a negative control sample containing resin but no
bait should be included. This control allows to separate the signal from the
specific protein–protein interaction from the nonspecific background binding
of prey to the resin.
3.2.1.3. Resin Equilibration
Mix resin by inverting the tube several times to obtain uniform suspension.
1. Dispense 50 μl of HaloLink resin into two 1.5-ml Eppendorf tubes (experimental
and control) and spin in centrifuge for 1 min at 800 ×g(see Note 3).
2. Carefully remove and discard the supernatant without disturbing the resin at the
bottom of the tube.
3. Add 400 μl of resin equilibration buffer, mix thoroughly by inverting the tube.
4. Centrifuge for 2 min at 800 ×gatroom temperature.
5. Carefully remove and discard the supernatant without disturbing the 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 50–100 μl of binding buffer (see Note 4).
Add co-factors, detergents or other reagents needed for specific protein–protein
interactions.
3.2.1.4. Binding the Bait
1. To the experimental resin sample, add 20 μl (or more if protein expression is low)
of cell lysate containing the HaloTag fusion protein.
2. To the negative control sample (resin without the bait), add 20 μl buffer or TnT®
T7 Quick Coupled Transcription/Translation mix without the DNA template.
3. Incubate by mixing on a tube rotator (see Note 6) for 30–60 min at room temperature
(incubate at 4°C if proteins are unstable, longer incubation time may be
required). Make sure resin does not settle to the bottom of the tube as that will
reduce efficiency of binding. During this incubation, you can set up TnT® T7
Quick Coupled Transcription/Translation for the prey, see Subheading 3.2.1.6.
4. Centrifuge for 2 min at 800 × g.
3.2.1.5. Washing
1. Add 1 ml of wash buffer containing 1 mg/ml BSA and mix thoroughly by inverting
the tube.
2. Centrifuge for 2 min at 800 × g. Discard the wash.
3. Repeat steps 1 and 2 two more times.
4. Add 1 ml of wash buffer containing 1 mg/ml BSA and mix thoroughly by inverting
the tube.
5. Incubate 5 min with occasional mixing.