Table 1.

Troubleshooting table.

Step	Problem	Causes	Suggestions
1.8	Low coupling efficiency of bait protein to beads	Sample buffer contains primary amines	• Avoid using buffers or reagents containing primary amines during initial protein purification • Remove any primary amines by buffer exchange
2.2, 2.4	Prey protein not soluble at high concentrations in binding buffer	Buffer conditions not compatible with protein	• Carefully screen protein solubility (adjust salt, pH, detergents) [6]. • Couple this protein to the beads
2.3	Protein precipitates during concentration with Amicon centrifugal filters	• Protein solubility • Spin to harsh • High local concentrations of protein on bottom of the filter	• See step 2.2 • Pre-soak filters in buffer containing 50% glycerol • Reduce centrifugation speed • Centrifuge in five minute increments, pipette protein sample up and down between spins
3.11	Prey protein binds to BSA beads	Protein is not stable	• Screen buffer conditions [6] • Ensure that beads are completely blocked • Block with ethanolamine instead of BSA
3.11	Prey protein is degraded	Protein is not stable	• Run gel of input to determine if the protein was degraded prior to assay • Shorten incubation time • Screen buffer conditions
3.11	Variation in Kd between experimental repeats	Binding efficiency varies due to temperature and buffer conditions	• Conduct all binding experiments at a constant temperature of 4°C • Use the same buffer for all repeats • Perform all experiments with the same buffer, especially when comparing the binding constants of wild-type versus mutant protein
3.11, 4.5, 5.2	Binding does not increase with increased protein concentration	Binding may be saturated	• Lower prey protein concentrations
3.11, 4.5, 5.2	Binding pattern erratic	Bead volume not even between different reaction tubes	• Carefully pipette equal amounts of beads • Slightly cut or trim pipette tips prior to pipetting beads
3.11, 4.3	SDS-PAGE gels expand, uneven running of gels	Beads loaded on gel	• Carefully avoid loading beads • Increase centrifugation time/speed prior to loading
3.11	No binding to bait protein beads	Concentration of bait or prey protein is too low	• Increase concentration of prey protein • Bind more bait. protein to beads
3.11	Bait protein does not bind	No interactions between bait and prey protein	• Check protein beads with a prey protein that is known to bind to bait (positive control)