Table 2 |.

Troubleshooting table

Step	Problem	Possible reason	Possible solution
1	Cell culture contamination	Improper aseptic technique	Regularly check cultures for bacterial or fungal contamination. This can be done by observing cultures under a light microscope. Commercial mycoplasma detection kits should be used regularly. To prevent contaminations, use strict aseptic technique
1A(iii)	Poor transfection efficiency	FreeStyle 293-F suspension are clustering	Vigorously vortex cells for 20–30 s to obtain cultures composed predominantly of single cells
1B(ii)	Cells appear excessively clumpy or stringy	Cells are in stationary phase or have low viability	Ensure cells are in early log-phase growth (1.5–3 × 105 cells/mL) to avoid long doubling times and low titers
			If cells have low viability, thaw out fresh cells and ensure viability is >95%
2	No detectable protein after purification	Low protein expression	Run a western blot to detect spike in the supernatant. Confirm that cells have >95% viability. We observe low yields with older cells
		Protein not properly eluted from resin	Boil purification resin in 1× Laemmli buffer, spin down debris, and run the supernatant on an SDS-PAGE gel. If protein is present, increase the concentration of d-desthiobiotin or imidazole. Multiple purification tags on trimeric spike increases affinity for the resin
14	No spike loading	Low/inaccurate concentration of spike or the protein has degraded	Increase/remeasure spike concentration. Run an SDS-PAGE to check if full-length spike is expressed
17	Nonspecific binding of analytes	Insufficient blocking reagent and detergent	Increase the concentration of BSA and detergent
37	Inaccurate or inconsistent plate readings	Precipitate may form over time after H2SO4 addition	Immediately read plate at 450 nm