Table 2 Troubleshooting table
From: Expression and characterization of SARS-CoV-2 spike proteins
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 |
