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Posted 3 months ago
You have four different proteins that you want to separate from each other.
Protein A has a pl of 6.8 and a molecular weight of 66,000 , Protein BB has a pl of 7.0 and a molecular weight to 125,000 , Protein CC has a pl of 6.1 and a molecular weight of 50,000 and Protein D has a pl of 10 and a molecular weight of 11,000 . What chromatography technique would you use and why? Can you predict the elution order
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Answer from Sia
Posted 3 months ago
Ion exchange chromatography followed by size exclusion chromatography
Chromatography Technique Choice: Ion exchange chromatography is suitable for separating proteins based on their pI (isoelectric point). Since the proteins have different pIs, they will bind to and elute from the ion exchange column at different pH values. After ion exchange chromatography, size exclusion chromatography can be used to separate the proteins based on their molecular weight
Predicting Elution Order for Ion Exchange Chromatography: The elution order will depend on the pH of the buffer used. If the pH is set between the pI of Protein A (6.8) and Protein B (7.0), Protein C (pI 6.1) will elute first, followed by Protein A, then Protein B. Protein D (pI 10) will elute last because it will not bind to the column at a pH below its pI
Predicting Elution Order for Size Exclusion Chromatography: After ion exchange chromatography, size exclusion chromatography will separate proteins based on size. The largest protein will elute first and the smallest last. Therefore, Protein B (125,000 Da) will elute first, followed by Protein A (66,000 Da), then Protein C (50,000 Da), and finally Protein D (11,000 Da)
Key Concept
Chromatography Techniques for Protein Separation
Ion exchange chromatography separates proteins based on charge (pI), and size exclusion chromatography separates them based on molecular weight.

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