TABLE 2.

Troubleshooting table.

Step	Problem	Possible reason	Solution
18	No bands are visible	Incorrect setup or there is no membrane-integrated product	Check the in-gel fluorescence setup with pure yeast-enhanced green fluorescent protein (yEGFP). Blue light is needed for best sensitivity. Bands should be visible down to 5 ng (ref. 2). If still no signal is visible for the experimental sample, increase exposure time. If GFP counts are low (<5,000 relative fluorescence units), the protein may not be expressed or was expressed but has degraded so that only ‘free GFP’ is left6 (see Fig. 3 for average fusion recovery into membranes from whole cells)
26	Incorrectly localized protein	Protein is incorrectly folded or has been re-routed to vacuoles for degradation	If a non-endoplasmic reticulum (ER) membrane protein is retained in the ER, it may be misfolded and unable to exit. Check localization under different culture condition, for example, growth at 20 °C after adding galactose. If it is located in vacuoles, then the protein may have been initially targeted correctly, but has been downregulated for degradation in vacuoles, a common problem for transporters in the presence of their substrates26 (e.g., Ura yeast transporter Fur4 when Ura is abundant27). Try using a specialized media, and/or a constitutive promoter, and/or different yeast strain28
31	Low breakage efficiency, that is, <70%	Sample is too viscous	If final OD600 was high (>7), cell suspension may need to be diluted further with cell resuspension buffer to achieve better breakage efficiency. If no improvement is detected, then increase breaking pressure to 40 kpsi (2.4 × 103 atm)
36	Solubilization efficiency <45%	Total protein concentration is too high	Make sure total protein concentration is at 3 mg ml−1. Solubilization efficiency can also be increased (typically) by 10–15% by the dilution of total protein to 1.5 mg m−1 in the same detergent concentration
38	Free-GFP peak in fluorescence size-exclusion chromatography trace	This is a sign of protein degradation	If free GFP-to-fusion peak is <10%, this is not usually problematic and one should carry on with the protocol, provided that the protein peak is monodisperse
			If free GFP-to-fusion peak is 20–50%, then try different detergent, and/or add glycerol, ligand, inhibitor, etc., to improve protein stability
			If free GFP-to-fusion peak >50%, severe proteolysis has occurred. Try parameters suggested for lowering the amount of protein degradation, but also consider modification of protein, for example, make N- and/or C-terminal truncation(s), adding N-terminal tag, for example, Flag-tag
	Only aggregation peak is apparent	Protein is unstable in this detergent	Try different detergents, additions to buffer (e.g., glycerol, ligand/inhibitor), or homologs to the protein of interest
43	Not all of the protein binds to the column as evident by GFP fluorescence measured in flow through	Saturation of Ni-NTA resin or detergent lowers binding	Typically binding efficiency is ~50–60% for GFP-fusions in yeast purifications. Efficiency may increase in a differ- ent detergent, but it is not predictable and often difficult to improve. The addition of more Ni-NTA resins or overnight batch incubation is worth testing, but in our hands such modifications affords only modest improvements (10–15%)
49	The solution appears slightly cloudy	A small fraction of tobacco etch virus (TEV) protease can precipitate during removal of glycerol by dialysis	Filter sample through 0.22-mM filter. To confirm precipitant is due to TEV protease, repeat dialysis with pure TEV only. In almost all such cases, TEV cleavage has been >98% complete, and the target protein has not aggregated
49	The appearance of a heavy precipitant is observed	The protein is unstable without GFP	To distinguish between general instability and instability due to removal of GFP-8His tag, repeat dialysis of eluate without adding His-tagged TEV protease. In either scenario (aggregation in general or aggregation due to GFP-8His removal), try dialysis in a buffer with a higher or lower pH (e.g., 6 and 8), add glycerol, and/or repeat in a different detergent, and/or add inhibitor, etc. Note, that GFP-fusions can still be completely functional as fusions