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Detection of Protein–Protein and Protein–DNA Interactions 199
to bind to the resin during an incubation step. This step is also known as
“pre-charging” of the resin. To this resin carrying the bait, a new protein
mixture containing the binding partner (prey) is added. The bait–prey complexes
are formed and then isolated from the protein mixture by resin precipitation
(pull-down). During this procedure, several washes are performed to remove
nonspecifically bound proteins. At the end, the prey protein is eluted and
analyzed on SDS–polyacrylamide gel electrophoresis (PAGE) gel or by mass
spectrometry.
In the second approach (see Subheading 3.2.2.), the bait and prey proteins
are first mixed and allowed to form complexes. Resin is added to the pre-formed
bait–prey complexes. Complexes bind to the resin during incubation and are
then isolated from the rest of the proteins by resin precipitation (spinning).
This approach may be closer to physiological conditions, but may be more
challenging because the concentration of the complexes may be rather low. In
the case of pre-charging of the resin, the local protein concentration (concentration
of the bait on the resin) is increased which increases the likelihood of
successful isolation of the prey.
It should be mentioned that in all the procedures described below (see
Subheadings 3.2. and 3.4.), it is important to perform control reactions. Control
reactions should contain all the components of the experimental sample, except
for the bait protein, for example, control would consist of the resin, mixed with
TnT® extract containing the prey. All the methods describe use of the control
in detail.
3.2.1. Detection of Protein–Protein Interactions by Pre-Binding
of HaloTag Fusion Protein (Bait) to HaloLink Resin
In the protocol below, we describe a “pull-down” method (12) for detection
of protein–protein interactions in which the bait protein is first immobilized
onto HaloLink resin. A protein mixture containing the binding partner (prey)
is added to the immobilized bait and is allowed to bind. Bait–prey complexes
are then isolated and prey protein is identified.
3.2.1.1. Phase 1
Synthesis of the HaloTag fusion protein (bait) in vitro using
TnT® T7 Quick Coupled Transcription/Translation system following
manufacturer protocol: During the incubation of the TnT® T7 Quick
Coupled Transcription/Translation reaction, equilibrate HaloLink resin (see
Subheading 3.2.1.2., steps 1–7). Keep resin resuspended in the binding buffer
until TnT® T7 Quick Coupled Transcription/Translation reaction is completed
(if needed resin can be kept in this buffer overnight at 4°C) (see Note 7).