For most proteins which of the following should be done to prevent denaturation
ID: 149119 • Letter: F
Question
For most proteins which of the following should be done to prevent denaturation of proteins during protein purification? Choose all correct answers. 1) Store purified proteins at low concentrations in glass or plastic. 2) Store protein solutions open to the air. 3) Include low concentrations of reducing agent. 4) Use ultrasonic stirrers to rapidly stir/whip protein solutions. 5) Include a buffer system in the solvent with pKa within 1 pH unit of protein's optimal pH 6. Include EDTA IN THE SOLVENT. 7. Store proteins on ice or at colder temperatures.
Explanation / Answer
Answer:
Case 1: Store purified proteins at low concentrations in glass or plastic (False/True)
Explanation: Dilute protein solutions (< 1 mg/ml) are more prone to inactivation and loss as a result of low-level binding to the storage vessel. Protein should be stored in clean, autoclaved glassware or polypropylene tubes to prevent microbial or proteolytic degradation.
Case 2: Store protein solutions open to the air (False)
Explanation: Storage at room temperature often leads to protein degradation and/or inactivity, commonly as a result of microbial growth.
Case 3: Include low concentrations of reducing agent (True)
Explanation: Reducing agents such a dithiothreitol (DTT) and 2-mercaptoethanol (2-ME) at final concentrations of 1-5 mM also help to maintain the protein in the reduced state by preventing oxidation of cysteines.
Case 4: Use ultrasonic stirrers to rapidly stir/whip protein solutions (True)
Explanation: Protein isolates with improved functional properties can be obtained using high intensity ultrasound technology.
Case 5: Include a buffer system in the solvent with pKa within 1 pH unit of protein's optimal pH (True)
Explanation: The protein stability also relies on the storage pH provided by the buffer. Hence it is essential to provide proper pH environment to ensure optimum stability.
Case 6: Include EDTA in the solvent (True)
Explanation: Metal chelators such as EDTA at a final concentration of 1-5 mM avoid metal-induced oxidation of –SH groups and helps to maintain the protein in a reduced state.
Case 7: Store proteins on ice or at colder temperatures (True)
Explanation: Generally, proteins are best stored at 4°C to prevent microbial or proteolytic degradation
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