Why did my Protein expression fail?
No detectable recombinant protein on gel / blot / activity after induction or transfection. Recombinant protein is produced but remains in the insoluble fraction / inclusion bodies. Product appears as fragments or disappears on storage / purification.
Playbooks
- Protein expression: no expressionNo detectable recombinant protein on gel / blot / activity after induction or transfection.
- Protein expression: insoluble proteinRecombinant protein is produced but remains in the insoluble fraction / inclusion bodies.
- Protein expression: degraded proteinProduct appears as fragments or disappears on storage / purification.
Calculators
FAQ — Protein expression
- No expression
- No detectable recombinant protein on gel / blot / activity after induction or transfection. Sequence-verify the expression clone (ORF + junctions) before another induction — Empty or frameshifted clones are the most common material miss. Run a validated positive-control construct side-by-side with fresh inducer — Separates method failure from construct failure in one experiment. Move the plasmid into a validated expression host (e.g. BL21(DE3) / HEK293T) — Promoter–host mismatch silently yields zero product.
- Insoluble protein
- Recombinant protein is produced but remains in the insoluble fraction / inclusion bodies. Induce at lower temperature (e.g. 16–20°C) for a longer time — Slower folding often rescues otherwise insoluble products. Add a solubility tag or co-express chaperones; re-test soluble fraction — Material redesign when temperature alone is insufficient. Screen lysis buffer pH / salt / detergent on small aliquots — Some “insoluble” pellets are buffer artifacts.
- Degraded protein
- Product appears as fragments or disappears on storage / purification. Lyse on ice with a fresh protease-inhibitor cocktail; minimise time to cold storage — Most degradation is preventable protease activity after lysis. Shorten boiling / use fresh reducing sample buffer for SDS-PAGE — Rules out gel artifacts before redesigning the ORF. Re-design construct (tags, domain boundaries) if inhibitors cannot preserve full length — Intrinsic instability needs a material change.