. Author manuscript; available in PMC: 2010 Oct 20.

Published in final edited form as: Nat Protoc. 2007;2(5):1145–1151. doi: 10.1038/nprot.2007.172

TABLE 3.

Troubleshooting table.

Step	Problem	Possible reason	Solution
	Tagged protein is not functional	Tag negatively interferes with protein's function	Test different constructs with the tag at the N or C terminus. Insert a flexible oligopeptide linker in between the tag and protein
7	Tagged protein is not present in the supernatant	Tagged protein is bound to membranes or insoluble particles	Modify the preparation of cell extract
8	Tagged protein is lost after filtration	Protein binds to the filter	Do not filter the protein extract (skip Step 8)
10–14	Degradation of proteins occurs	HeLa cells were grown in plates and harvested by trypsinization	Avoid using trypsin, harvest cells by scraping
14	Degradation of proteins occurs	Presence of proteases in the sample	Add protease inhibitors into TEV cleavage buffer (1 tablet of Complete Mini Protease Inhibitor (Roche) per 20 ml, 0.5 mM PMSF)