Table 2.

Troubleshooting

Problem	Possible cause	Solution
No colonies on the transformation plates	Wrong antibiotics in the plate Bad competent cells Wrong plasmid	Make fresh plates Try fresh cells Sequence the plasmid
No cell growth in the flask	Too many antibiotics Failed shaker incubator	Add accurate amount of antibiotics Check incubator
No protein expression	Glucose inhibition Bad IPTG	Remove glucose before induction Make fresh IPTG
Low yield	Insufficient Polymyxin B	Make fresh stocks and add enough amount
	Bad HisTrap column	Try new column
Low purity	Not enough washing stringency	Increase washing stringency by increasing imidazole concentration (less then 50 mM) in the washing step
No ELISA signal	Bad TMB substrate or secondary antibodies	Use positive controls and test the secondary antibody with the TMB substrate directly
High background on ELISA	Insufficient blocking or washing	Increase blocking time or Including milk or BSA in the blocking buffer Wash more