Stabilizing proteins after yeast cell disruption: Which additive(s) can be included to protect protein structure and activity immediately after mechanical lysis?

Introduction / Context:
Upon disrupting yeast or other cells, endogenous proteases, shear stress, and ionic imbalances threaten protein integrity. Appropriate additives can stabilize targets and improve downstream purification yield.

Given Data / Assumptions:

• Lysis releases proteases and nucleases.
• Protein aggregation is promoted by inappropriate salt or pH.
• Some enzymes require ligands (for example, nucleotides) for stability.

Concept / Approach:
Adding salt such as NaCl tunes ionic strength, minimizing nonspecific electrostatic interactions. Protease inhibitors (for example, PMSF, leupeptin cocktails) suppress proteolysis. Ligands like AMP or cofactors can stabilize the native conformation of allosteric or nucleotide-binding proteins, reducing denaturation and loss of activity.

Step-by-Step Solution:
Supplement lysis buffer with NaCl (commonly 100–300 mM).Add a broad-spectrum protease inhibitor mix at recommended concentration.Include stabilizing ligands (AMP, ATP analogs, metal ions) if compatible with the target.

Verification / Alternative check:
Compare activity recoveries with and without additives; improved activity and reduced degradation bands on SDS-PAGE confirm protection.

Why Other Options Are Wrong:

• Single additive only: Often insufficient; multiple stressors exist after lysis.
• Detergent only: Useful for membranes but does not address proteolysis or charge screening alone.

Common Pitfalls:
Forgetting EDTA/EGTA when metalloproteases are present; adding ligands that interfere with downstream binding (for example, affinity resins).

Final Answer:
All of these.