Applications of GST Pulldown

1
Applications of GST Pulldown

• Observe protein-protein interactions
• Assay binding pathway for a protein complex
• Identify specific binding sequences

2
Critical parameters

• Concentration of fusion protein
• Too much fusion protein leads to non-specific
  interactions
• Concentration of target protein
• Too much target protein makes it difficult to
  estimate relative binding to fusion protein

3
False Positives

• Non-specific interactions of target protein with
  GST alone
• Non-specific interactions of target protein with
  the beads

4
Does the target protein interact with GST alone?

• Target protein Beads with bound GST

5
Does the target protein interact with the Beads?

• Beads target protein
• Important when target protein will be collected
  by methods other than elution with GSH buffer
• GSH elution buffer will only disrupt GSH-GST
  interactions

6
Interpreting Negative Results

• Suboptimal chemical conditions
• Steric hindrance caused by fusion
• Fusion protein not modified in E. coli
• Other factors required for interaction
• Fusion protein in inclusion bodies
• Degradation of fusion protein
• No interaction

7
Troubleshooting negative results

• Steric hindrance
• Move GST sequence to N-terminus
• Fuse test protein to GST and use initial bait
  protein as test protein .
• Use a different tag
• Not modified in E. coli
• Express fusion in yeast or mammalian cells
• High levels of glutathione in eukaryotes
  may compete with the beads for GST binding sites
  

8
Troubleshooting negative results

• Other factors required for interaction
• Repeat the assay in the presence of a cellular
  extract
• Fusion protein in inclusion bodies
• resuspend and treat pellets with reagents that
  promote proper folding.

9
Troubleshooting negative results

• Degradation of fusion protein
• use protease deficient cells such as BL21
• Add protease inhibitors like PMSF
  (phenylmethansulfonyl fluoride)
• Reduce final concentration of inducer
• Induce after reaching a higher OD
• Induce for a shorter period

10
No interaction