Step	Problem	Possible reason	Possible solution
17	Only asmall fraction of GB1 binds to the StrepTRap HP column	The sample injection is too fast	Inject the sample to the column slowly (less than 1 mL/min); connect a few columns together; clean the columns after each use
22	The efficiency of the reaction between TriNTA and SMCC is low	Incorrect buffer conditions	Make fresh sodium carbonate buffer (pH 8.0). The pH of sodium carbonate can increase to 10 after about one week
29	The efficiency of the reaction between SMCC-TriNTA and GB1 is low	GB1 is not completely reduced; GB1 is oxidized by air during the reaction; the amount of SMCC-TriNTA is not enough	Make sure that the GB1 is completely reduced before the reaction; degas the buffer; use a higher excess of SMCC-TriNTA
43	The binding between the TriNTA-GB1 and Foldon resin is weak	The resin is not saturated by Foldon; the incubation time is too short; residual imidazole is present in the buffer	Use at least 20 mg of Foldon per 150 mg of dry resin; incubate the TriNTA-GB1 and the Foldon resin for at least 30 minutes; make sure that no imidazole is present on the resin nor in the TriNTA-GB1 buffer; repeat the purification procedure a few times to achieve high TriNTA-GB1 purity
52	The efficiency of the cross-linking reaction is low	The His6-tags of the TM protein are too close in space; the protein concentration is low; the cross-linker is too short	When multiple His6-tags are too close in space, the binding stoichiometry between His6-tag and TriNTA may not be 1:1 anymore. Introduce the His6-tag based on the protein structure so that the His6-tags are fairly distant; add charged residues in the proximity of the His6-tag to introduce electrostatic repulsion near the latter; use concentrated protein; use longer cross-linkers