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Question
Biology
Posted 6 months ago

What would likely happen if your elution buffer contained 5mM5 \mathrm{mM} imidazole instead of 500mM500 \mathrm{mM} ?
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Answer from Sia
Posted 6 months ago
Answer
If your elution buffer contained 5mM5 \mathrm{mM} imidazole instead of 500mM500 \mathrm{mM}, the likely outcome would be that the target protein would not elute efficiently from the affinity column.
Solution
a
Imidazole concentration: Imidazole competes with histidine residues on the target protein for binding to the nickel ions on the affinity column. A lower concentration of imidazole (5mM5 \mathrm{mM}) would not be sufficient to displace the target protein from the column
b
Elution efficiency: At 500mM500 \mathrm{mM} imidazole, the competition is strong enough to effectively elute the target protein. At 5mM5 \mathrm{mM}, the target protein would remain bound to the column, resulting in poor or no elution
Key Concept
Imidazole concentration in elution buffer
Explanation
The concentration of imidazole in the elution buffer is critical for the efficient elution of the target protein from an affinity column. Lower concentrations may not effectively compete with the histidine residues, leading to poor elution.

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