Table 2. Troubleshooting commonly encountered problems.

Problem	Possible cause	Potential solution
Low protein yield	Insufficient IPTG induction Culture time insufficient or prolonged Suboptimal cell lysis Protein degradation during purification Poor protein-bead binding Poor protein elution	Optimize IPTG concentration and timing Optimize culture time Increase sonication intensity, frequency; use French press Optimize protease inhibitors, keep solutions on ice whenever possible, avoid prolonged purifications Run SDS-PAGE of beads to determine if protein bound at each purification stage; verify pH of column buffers; increase loading time (incubate beads with lysate overnight at 4 °C) Run SDS-PAGE of beads and supernatants to determine if protein bound at each purification stage; increase elution gradient; verify pH of elution buffers
Low protein purity	Protein degradation during purification process Pooling impure fractions Insufficient bead washing Suboptimal imidazole gradient Additional purification needed	Optimize protease inhibitors; keep solutions on ice whenever possible; avoid prolonged purifications Run SDS-PAGE, pool purest fractions Increase washing volumes Optimize imidazole gradient Perform gel filtration
Poor signal	Insufficient 13C-leucine incorporation Insufficient protein reporter present Suboptimal instrument settings Suboptimal pulse sequences Protein denaturation during reaction, sample queue, or sample acquisition	Optimize timing of labeled precursors relative to IPTG induction; optimize concentration of labeled precursors Verify protein concentration; increase La antigen concentration Check NMR spectrometer shimming, other settings Optimize pulse sequences Lower temperature of reaction
Positive control inactive	Compound degradation Suboptimal reaction conditions Compound precipitation DTT contamination in DTT-free reactions	Verify compound identify and purity; verify compound stability in assay buffer Verify reaction temperatures and duration; increase reaction temperature or duration Lower compound concentration; increase temperature during sample queue Increase dialysis timing and/or number of buffer changes
Negative control active	Compound degradation/impurity Protein denaturation	Verify compound identify and purity; verify compound stability in assay buffer Reduce time between reaction and NMR spectra acquisition; verify proper sample handling including temperature