Overview
This lecture covers ion exchange chromatography, a widely used protein purification technique that separates proteins based on their net charge differences and their isoelectric point (pI).
Charge and Isoelectric Point (pI)
- Proteins have an isoelectric point (pI), the pH at which the net charge is zero.
- At pH < pI, proteins are positively charged (net positive).
- At pH > pI, proteins are negatively charged (net negative).
- Proteins are least soluble at their pI and tend to aggregate.
Types of Ion Exchange Chromatography
- Two types: cation exchange (binds positive ions) and anion exchange (binds negative ions).
- Cation exchange resin is negatively charged and binds cations.
- Anion exchange resin is positively charged and binds anions.
- Common resins: DEAE cellulose (anion exchanger, positive resin), CM cellulose (cation exchanger, negative resin).
Procedure and Mechanism
- Pour column with resin and install a filter or cotton to retain resin.
- Wash column with buffer matching protein’s buffer.
- Load protein mixture to separate the protein of interest.
- Nonbinding proteins (same charge as resin) flow through and are collected.
- Bound proteins are eluted by increasing salt concentration or changing pH (salt gradient is more common).
- Weakly bound proteins elute at lower salt; strongly bound proteins at higher salt.
Detection and Fraction Collection
- Eluted proteins are collected in sequential fractions (tubes).
- UV absorbance at 280 nm is commonly used to detect protein-containing fractions.
- Additional assays may be required to identify fractions containing the protein/enzyme of interest.
- Colored proteins can be visually observed to aid collection.
Further Purification and Analysis
- Eluted fractions often contain high salt and need further processing (e.g., dialysis or gel filtration) to remove salts.
- Check protein purity using techniques like gel electrophoresis.
- Ion exchange chromatography is often combined with other purification methods.
Key Terms & Definitions
- Isoelectric Point (pI) — pH where a protein's net charge is zero.
- Cation Exchange Chromatography — uses negatively charged resin to bind positively charged proteins.
- Anion Exchange Chromatography — uses positively charged resin to bind negatively charged proteins.
- Resin — solid phase in the column that interacts with proteins based on charge.
- Elution — process of releasing bound proteins from the resin, typically by increasing salt concentration.
Action Items / Next Steps
- Look up additional types of ion exchange resins (e.g., on Bio-Rad's website).
- Review the prices of ion exchange resins for context.
- Practice identifying whether a protein should use cation or anion exchange based on its pI and buffer pH.