Troubleshooting

Problem	Potential Cause and Solution
The protein expresses but is only in the pellet after lysis	It is wise to first confirm expression and presence of the fusion protein in the pellet through Western analysis. If confirmed, this suggests that the protein is insoluble. There are several potential causes and solutions to this. (1) The lysis buffer could be incompatible with the protein, check the composition of the buffer especially the pH and salt concentration and alter to increase solubility. (2) The cells were not lysed, try relysing, another lysis method or a combination oflysis methods to increase lysis efficiency. (3) The protein misfolds during expression. This can usually be addressed by slowing down the rate of expression. Lower the concentration of IPTG for induction and/or lower induction temperature
The protein does not bind to the resin	The potential cause ofthis depends on the tag. (1) Confirm that the binding buffer is compatible with the resin. (2) If using a structured fusion tag, it may indicate that the tag unfolds, which could occur either during expression or lysis. Try different induction conditions described above for solubility. Or try altering the lysis method to a less harsh method. (3) The resinbinding site on the tag may be occluded by the domain ofinterest. Try altering the linker between the fusion tag and domain, switching termini for placement of the tag, or switching tags
There is no cleavage reaction	The cleavage site may be occluded. (1) Try eluting off the resin first and cleaving in solution. (2) Try altering the linker between the fusion tag and protein, switching the termini that the tag is on, or switching tags
The protein cleaves but the tag-free domain does not elute from the resin	It is likely that the cleaved domain is either sticking to the resin or precipitating after cleavage. (1) Try eluting with a higher salt buffer to reduce sticking. (2) Try cleaving after eluting the fusion protein from the resin. (3) Alter cleavage conditions to promote solubility ofthe tag-free domain. (4) Switch tags