Protocols and Applications Guide (US Letter Size) - Promega

||||||||||| 11Protein Purification and Analysis
Phase 1
In vitro Prey Protein Synthesis
(TNT ® T7 System)
P P P
P
M GST
GST
Wash
Elute
+
prey protein
detected
P
P
Phase 3
Prey Capture
Phase 2
Immobilization of Bait (GST-fusion)
Protein onto MagneGST Particles
Experimental
Control
(GST-Fusion) (GST)
M GST
M GST
Wash
MElute
GST
Bait
P
P
Bacterial
Cells
4402MA12_3A
Lysis
Binding
M GST
Figure 11.9. Schematic diagram of the MagneGST Pull-Down System protocol. P = prey protein, M = MagneGST Particle.
2. Add 200µl of MagneGST Cell Lysis Reagent to each
fresh or frozen cell pellet. Resuspend the cell pellet at
room temperature (20–25°C) by pipetting or gentle
mixing.
3. Add 2µl of RQ1 RNase-Free DNase.
Note: Addition of DNase reduces viscosity and can
increase the purity of GST-fusion proteins. Up to 5µl
of RQ1 RNase-Free DNase can be added to reduce
viscosity. The DNase can be omitted, if desired.
4. Incubate the cell suspension at room temperature for
20–30 minutes on a rotating platform or shaker. During
this incubation, begin the particle equilibration
procedure.
Equilibrate Particles
1. Thoroughly resuspend the MagneGST Particles by
inverting the bottle several times to obtain a uniform
suspension.
Protocols & Applications Guide
www.promega.com
rev. 3/09
2. Pipet 20µl of MagneGST Particles into a 1.5ml tube.
3. Place the tube in the magnetic stand, and allow the
MagneGST Particles to be captured by the magnet.
Magnetic capture will typically occur within a few
seconds.
4. Carefully remove and discard the supernatant.
5. Remove the tube from the magnetic stand. Add 250µl
of MagneGST Binding/Wash Buffer to the particles,
and resuspend by pipetting or inverting.
6. Repeat Steps 3–5 two more times for a total of three
washes.
Bind Protein
1. After the final wash, resuspend the particles in 100µl
of MagneGST Binding/Wash Buffer.
Note: Adding up to 1% BSA may reduce nonspecific
binding and potential problems with background.
PROTOCOLS & APPLICATIONS GUIDE 11-17