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Detection of Protein–Protein and Protein–DNA Interactions 197
Vectors designed for expression in mammalian cells or in the in vitro protein
expression systems. Flexi® Vectors provide a rapid, highly reliable system for
cloning and transfer of coding regions between vectors containing various tags
and expression options.
3. Methods
This section provides guidelines on how to immobilize HaloTag fusion
proteins onto HaloLink resin (see Fig. 2). Immobilized proteins can then be
evaluated for in vitro protein–protein interactions (see Subheadings 3.2.1. and
3.2.2.), in vivo protein–protein interactions (see Subheading 3.3.), protein–DNA
interactions (see Subheading 3.4.), enzymatic activity (see Subheading 3.5.) and
for isolation of protein fused to HaloTag by proteolytic cleavage of the fusion
protein bound to the resin (see Subheading 3.6.)(see Fig. 2).
3.1. General Protocol for Immobilization of HaloTag Fusion
Proteins onto the HaloLink Resin
The protocol below is optimized for binding of proteins expressed in the
in vitro expression systems (see Fig. 2). We used TnT® T7 Quick Coupled
Transcription/Translation System (cat. no. L1170, Promega). Other in vitro
expression systems can be used. These reactions are typically 50 μl, which may
be sufficient for more than one immobilization reaction. This protocol can also
be used for immobilization of proteins expressed in vivo in mammalian cells.
If mammalian expression systems are used optimize amounts of resin and cells,
follow the steps described in Subheading 3.3 through phase 3 washing as a
guideline. Different lysis conditions can be used, see also Subheading 3.4.
step 10.
3.1.1. Phase 1
Synthesis of the HaloTag fusion protein 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 3.1.2., 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).
3.1.2. Phase 2: Resin Equilibration
Mix resin by inverting the tube several times to obtain uniform suspension.
1. Dispense 50 μl of HaloLink resin into 1.5-ml Eppendorf tube and spin in
centrifuge for 1 min at 800 ×g(see Note 3).