GST-Trap M

Bead size 0.5 - 1 µm

• Robust and versatile tool for biochemical analyses of GST-fusion proteins
• Short incubation times (5 - 30 min)
• Low unspecific binding
• Quantitative isolation of fusion proteins and transiently bound factors from cell extracts or organelles
• No contaminating heavy and light chains of conventional antibodies

Suggested buffer composition

• Lysis buffer: 10 mM Tris/Cl pH 7.5, 150 mM NaCl
• Dilution buffer: 10 mM Tris/Cl pH 7.5, 150 mM NaCl
• Wash buffer: 10 mM Tris/Cl pH 7.5, 150 mM NaCl
• Elution buffer: 200 mM glycine pH 2.5

• 1. For one immunoprecipitation reaction resuspend cell pellet of 1 mL bacteria culture expressing GST in 100 - 200 µL lysis buffer by pipetting.
  optional: add 1 mM PMSF and 0.1 mg/ml lysozyme to lysis buffer
• 2. Rotate tube for 1h at 4°C.
• 3. Sonify cell pellet to disrupt bacterial membrane.
• 4. Spin cell lysate at 20.000x g for 5 -10 minutes at 4°C.
• 5. Transfer supernatant to a pre-cooled tube. Adjust volume with dilution buffer to 500 µL. Discard pellet.For immunoblot analysis dilute 30 µL cell lysate with 10 µL 4x SDS-sample buffer(-> refer to as input).
• 6. Equilibrate GST-Trap_M beads in dilution buffer. Resuspend magnetic particles by vortexing and transfer calculated volume (20 - 30 µL) in a new reaction cup with 250 µL ice cold dilution buffer. Magnetically separate until supernatant is clear and wash twice with 250 µL of cold dilution buffer.
• 7. Add cell lysate to equilibrated GST-Trap_M beads and incubate the GST-Trap_M beads with the cell lysate under constant mixing for 10 min – 2 h at room temperature or 4°C.
  note: during incubation of protein sample with the GST-Trap_M the final concentration of detergents should not exceed 0.2% to avoid unspecific binding to the matrix
• 8. Magnetically separate until supernatant is clear. For western blot analysis dilute 30 µL supernatant with 10 µL 2x SDS-sample buffer (-> refer to as non-bound). Discard remaining supernatant
• 9. Wash beads two times with 500 µL ice cold wash buffer.
  optional: increase salt concentration in the second washing step up to 500 mM
• 10. Resuspend GST-Trap_M beads in 100 µL 2x SDS-Sample buffer or go to step 11.
• 11. Boil resuspended beads for 10 minutes at 95°C to dissociate the immunocomplexes from the beads. The beads can be collected by centrifugation at 2.500x g for 2 minutes at 4°C and SDS-PAGE is performed with the supernatant (-> refer to as bound).
• 12. optional: elute bound proteins by adding 50 µL 0.2 M glycine pH 2.5 (incubation time: 30 sec under constant mixing) followed by centrifugation. Transfer the supernatant to a fresh cup and add 5 µL 1M Tris base (pH 10.4) for neutralization. To increase elution efficiency this step can be repeated.