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Detection of Protein–Protein and Protein–DNA Interactions 201
6. Centrifuge for 2 min at 800 × g. Discard the wash.
7. Repeat steps 4–6 one more time.
8. Resuspend resin in 100 μl of wash buffer containing 1 mg/ml BSA (keep the resin
with immobilized bait at 4°C until prey synthesis is finished).
3.2.1.6. Phase 3
Synthesis of the prey in vitro using TnT® T7 Quick Coupled Transcription/
Translation System following manufacturer protocol: Label the prey protein by
adding [ 35 S] methionine (2 μl) (1000 Ci/mmol at 10 mCi/ml) or FluoroTect
Green in vitro Translation Labeling System (cat. no. L5001, Promega) into the
in vitro TnT® T7 Quick Coupled Transcription/Translation reaction; follow
instructions given by manufacturer (see Notes 7 and 8).
3.2.1.7. Phase 4: Capture and Analysis of Prey Protein Capture
1. Add 20 μl of the TnT® T7 Quick Coupled Transcription/Translation reaction from
phase 3 to the resin carrying HaloTag fusion protein and to the negative control
resin (no bait) prepared in phase 2.
2. Incubate by mixing on a tube rotator (see Notes 6 and 9) for 1 h at room
temperature. Make sure resin does not settle to the bottom of the tube as that will
reduce efficiency of binding.
3. Centrifuge for 3 min at 800 × g. Discard the supernatant.
3.2.1.8. Washing
Stability of different protein–protein interactions is protein pair specific and
depends on the affinity of interaction. If interaction is not very stable, the
washing conditions used for these protein pairs may have to be optimized,
for example, change the number and volume of washes. However, insufficient
washing may result in detection of nonspecific interactions.
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 for 5 min with occasional mixing.
6. Centrifuge for 2 min at 800 × g. Discard the wash.
7. Repeat steps 4–6 one more time.
3.2.1.9. Elution
1. Add 20 μl of 1× SDS loading buffer (for composition see Subheading 2; 0.5 or
0.25 × elution buffer can also be used).
2. Incubate 2–5 min at 90°C (see Note 10).
3. Remove supernatant and load on a SDS–PAGE gel for analysis.