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Proteins can be separated according to their net charge using the potent protein purification method known as ion exchange chromatography (IEC).
The variations in net charge determine the protein separation of IEC. There are charged groups, either cationic or anionic, bonded to a solid support in the stationary phase. Proteins with opposite net charges to those in the stationary phase are retained, whereas those with similar charges flow through quickly.
Adjust pH and ionic strength to optimize protein stability and solubility during sample preparation for interaction with the ion-exchange resin during chromatography.
Prior to sample application, equilibrate the ion-exchange column using buffer at pH conditions compatible for protein binding as well as ionic strength conditions.
Put prepared protein sample onto ion-exchange column in conditions promoting binding of target protein to resin; monitor elution absorbance or conductivity for tracking protein elution
Use buffer solution for washing out unbound proteins and other contaminants from the column while retaining target proteins in its resin.
Altering buffer conditions such as pH or ionic strength cause elution of bound proteins leading to disruption of interactions between these molecules and resins thereby releasing bound target protein collect fractions containing desired protein based on absorbance or conductivity measurements.
Assess purity and yield by analyzing eluted fractions using techniques like SDS-PAGE or spectroscopy Pool together only those fractions containing purer forms that have a target protein for further downstream applications.
After every use, wash with proper solutions that remove bound proteins and regenerate resin for subsequent purifications
During purification process, such as poor binding, excessive column backpressure or protein aggregates, monitor flow rate, column pressure and elution profile in chromatography parameters.
While retaining a consistent purification performance and protein yield it is necessary to scale up the purification process when dealing with larger sample volumes.
Understanding the principles and best practices of ion exchange chromatography will enable you to purify proteins with high yield and purity for various downstream applications in biochemistry, biotechnology and pharmaceutical research.
