5- using ion-exchange chromatography to separate a mixture of proteins

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4 Terms

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Different types of chromatography exploit different protein differences

  • Size (molecular weight)

    • Gel filtration

  • Isoelectric point

    • Ion-exchange

  • Solubility

    • Hydrophobic interaction

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Ion-exchange

  • Based on charge interaction

  • Ion-exchanger column = positively charged

    • E.g/ diethylaminoethyl

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How proteins are detected in ion-exchange

  • Use easily detectable proteins

    • Methaemolglobin - absorbs light at 410nm

    • Green fluorescent protein - absorbs at 490nm

  • MetHb passes through low ionic strength buffer - pH = 7.2

  • GFP binds to DEAE cellulose - pH 5.8

    • Elute GFP with high ionic strength infer

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Percentage yield

(total amount of protein recovered from the column)/(total amount of proteins applied to the column)