Hi all, I want to show the degradation of a large bio molecule after exposure to a caustic cleaning agent via SDS-PAGE. Would I use reduced or non-reduced conditions for that? First thought was to run it native as the protein should be already degraded but without using any DTT or Mercapto the peptides that are still intact wouldn’t be unfolded and I’m not sure whether they would run that well. I would probably have to dialyse as well to get rid of the cleaning agent? Any suggestions welcome. Thank you!
There is no requirement to demonstrate the degraded bio-molecule exists. Just demonstrate that it is cleaned from the surface.
You can use Bradford’s reagent or native SDS-PAGE.
Remember, ‘Botox’ is an analog of ‘Clostridium botulinum neurotoxin’ and either must use the ADE (or PDE) in carryover calculations OR be manufactured in a dedicated facility in order to prevent cross-contamination
Thank you! What is the reason to use native instead of reduced you think?
From my past experience (with the pituitary hormone Oxytocin) the reduced form is inactive. If your protein contains a Cysteine bridge it will be closed under reduced conditions (and unavailable).
Therefore, it is not part of the safety (toxicity) profile used in cleaning validation assessments.