Protein expression: degraded protein

1. Ranked causes

   1. Protease activity during lysis / purification (inhibitors missing or expired)Fresh protease-inhibitor cocktail preserves the full-length band.
   2. Intrinsic instability of the construct (missing domains / wrong isoform)Degradation pattern reproduces across fresh preps with inhibitors present.
   3. Samples held too long at warm temperature after lysisImmediate cold processing stops fragmentation.
   4. Gel / blot sample-buffer artifacts (boil too long, wrong reducing agent)Native or milder loading shows intact product.

2. Recovery steps

   1. Lyse on ice with a fresh protease-inhibitor cocktail; minimise time to cold storageMost degradation is preventable protease activity after lysis.
   2. Shorten boiling / use fresh reducing sample buffer for SDS-PAGERules out gel artifacts before redesigning the ORF.
   3. Re-design construct (tags, domain boundaries) if inhibitors cannot preserve full lengthIntrinsic instability needs a material change.

3. Controls first

   • Did a stable positive-control protein stay intact through the same lysis and gel?If yes: This construct’s proteases or intrinsic instability — not a global buffer failure.
   • Does fresh protease-inhibitor cocktail preserve the full-length band?If yes: Protease control first — do not redesign the ORF yet.

Diagnose your run

Opens Diagnostics capture with this first-party playbook behind it. Sign in if the workspace asks.