Protein expression: no expression

1. Ranked causes

   1. Wrong or frame-shifted construct / empty vector cloneSequencing or diagnostic digest disagrees with the intended ORF.
   2. Induction / transfection conditions never turned expression onPositive-control construct expresses in the same batch; IPTG/serum timing off.
   3. Host strain / cell line incompatible with the promoter systemSame plasmid expresses in a validated expression host but not this one.
   4. Detection antibody / assay cannot see the tag or isoformTag-independent stain or activity assay still blank — or only the blot is blank.

2. Recovery steps

   1. Sequence-verify the expression clone (ORF + junctions) before another inductionEmpty or frameshifted clones are the most common material miss.
   2. Run a validated positive-control construct side-by-side with fresh inducerSeparates method failure from construct failure in one experiment.
   3. Move the plasmid into a validated expression host (e.g. BL21(DE3) / HEK293T)Promoter–host mismatch silently yields zero product.

3. Controls first

   • Did a positive-control construct express in the same induction / transfection batch?If yes: This construct, host compatibility, or detection — not a global method failure.
   • Did a positive-control construct express in the same induction / transfection batch?If no: Fix induction/transfection reagents and timing before redesigning the ORF.

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