Step	Problem	Possible reason	Solution
6	Too much precipitant	Chlorosulfonic acid was old or too much moisture was introduced into the reaction, leading to hydrolysis of chlorosulfonic acid	Use fresh, dry reagents and ensure dry conditions for synthesis. Purity is not affected as the unreacted material is removed through filtration so this problem is tolerable.
18 and 19	Amber mutant yields protein in the absence of sulfotyrosine	Purification is problematic or there is a mutation in the expression vector	Background incorporation of natural amino acids in the absence of sulfotyrosine by our synthetases has never been detected in our experiments. Therefore, check that the expression vector contains the desired amber mutant, and that purification has removed all truncated protein that may complicate analysis.
18 and 20	Yield of sulfotyrosine-containing protein is undetectable, but yield of full-length protein with tyrosine encoded in the stead of sulfotyrosine is high	Mutation in a tRNA cassette promoter that disables expression of tRNA	Sequence the synthetase plasmid and if there is a mutation in the tRNA region, repeat expression from step 13, ensuring that the clone picked is one of the small majority colonies, not a rare outlier large colony.
18 and 20	Yield of sulfotyrosine-containing protein is consistently very low compared to yield of full-length protein with tyrosine encoded in the stead of sulfotyrosine	Suppression of amber codon is inefficient	Choose another site for sulfotyrosine incorporation if possible. If not, silently mutate the codons before and after the TAG codon as codon context of TAG has been shown to correlate to expression efficiency.
18 and 20	Yield of sulfotyrosine-containing protein and yield of full-length protein with tyrosine encoded instead of sulfotyrosine are both consistently low	Suboptimal expression conditions and temperature	Make sure saturation is achieved after 6–8 hours from induction. If it is not, the protein being expressed is likely toxic. In this case, it will be necessary to induce at a higher OD and express at lower temperatures. For optimal expression, saturation should occur at an OD595 greater than 2.0 in rich media. (In fact, we have observed, in some cases, a saturated OD595 of ~14.0 using TB growth media.)