Overview
This lecture covers ion exchange chromatography as a protein purification technique, focusing on how protein charge varies with pH and application of this knowledge to separate proteins by charge.
Protein Charge and Isoelectric Point (pI)
- Proteins have an isoelectric point (pI), the pH where the net protein charge is zero.
- At the pI, proteins are least soluble and prone to aggregation.
- Below the pI (pH < pI), proteins have a net positive charge (more protonated amino groups).
- Above the pI (pH > pI), proteins have a net negative charge (more deprotonated carboxylate and amino groups).
- Changing pH alters the charge state of proteins due to their amino acid composition.
Ion Exchange Chromatography Principles
- Separates proteins based on charge differences using a charged resin.
- Two types: cation exchange (binds positive ions) and anion exchange (binds negative ions).
- Cation exchange columns have negatively charged resin to bind positively charged proteins.
- Anion exchange columns have positively charged resin to bind negatively charged proteins.
Common Resins
- DEAE cellulose: positively charged resin, used for anion exchange.
- CM cellulose: negatively charged resin, used for cation exchange.
Chromatography Procedure
- Pour column with chosen resin and filter material to keep resin in place.
- Wash column with buffer that matches protein solution.
- Load protein mixture onto the column; unwanted proteins (not binding) are found in flow-through.
- Wash column to remove unbound or weakly bound contaminants.
- Elute bound proteins using either a pH or salt gradient; salt gradient is more common to avoid protein precipitation.
- Increase salt concentration gradually to release proteins based on strength of their interaction with the resin.
Protein Detection and Collection
- Collected in separate fractions as proteins elute from the column.
- UV absorbance at 280 nm is commonly used to detect proteins in fractions.
- Additional assays may be required to identify fractions containing the target protein.
Post-Chromatography Steps
- High salt concentration in fractions often removed by dialysis or gel filtration.
- Further purification or purity assessment may be performed, such as gel electrophoresis.
Key Terms & Definitions
- Isoelectric point (pI) — the pH at which a protein’s net charge is zero.
- Cation exchange chromatography — separation method binding positively charged proteins via negative resin.
- Anion exchange chromatography — separation method binding negatively charged proteins via positive resin.
- Resin — the charged material used in the column to bind proteins of opposite charge.
- Elution — the process of releasing bound proteins from the resin.
- Flow-through — solution containing proteins that did not bind to the resin.
Action Items / Next Steps
- Look up and familiarize yourself with additional ion exchange resins (e.g., via Bio-Rad website).
- Prepare for lab protocols related to ion exchange chromatography.
- Review detection methods such as UV spectrophotometry at 280 nm.
- Study post-chromatography cleanup methods (dialysis, gel filtration).