Overview
This lecture covers strategies for protein purification and characterization, including the theoretical basis of several key analytical techniques and practical methods for quantifying and analyzing proteins.
Protein Purification: Principles and Planning
- Purifying soluble proteins is easier and yields better data than insoluble proteins (e.g., collagen, keratin).
- Pure proteins are necessary to avoid interference from contaminants in assays.
- Proteins are often drug targets, requiring pure samples for inhibitor testing.
- First, choose a detection assay for your protein (e.g., enzyme assay, colorimetric methods).
Methods for Protein Detection and Quantification
- Protein concentration can be measured using methods like the Lowry assay, Bradford assay, BCA assay, or absorbance at 280 nm (A280).
- Microtiter plate readers allow high-throughput enzyme and protein assays.
Protein Purification Techniques
- Differential centrifugation separates cellular components to enrich for target proteins.
- Dialysis removes small molecules or salts from protein solutions using semi-permeable membranes.
- Proteins are least soluble at their isoelectric point (pI) due to minimal net charge and reduced repulsion, causing aggregation.
Chromatographic Methods
- Ion exchange chromatography separates proteins by charge; anion exchangers bind negatively charged proteins.
- Gel filtration (size exclusion) separates proteins by size; large proteins elute first.
- Affinity chromatography uses specific biological interactions for purification, such as antibody-antigen binding.
Combining Purification Techniques
- Multiple steps (e.g., ion exchange, gel filtration, affinity chromatography) may be needed to achieve high purity and yield.
- Recovery, specific activity, and purification fold are used to track purification progress.
SDS-PAGE and Electrophoresis
- SDS-PAGE separates proteins by size using SDS to impart a uniform charge-to-mass ratio and a polyacrylamide gel matrix.
- Larger proteins migrate more slowly; bands can be visualized and compared to standards.
- Two-dimensional electrophoresis (isoelectric focusing + SDS-PAGE) separates proteins by pI and size.
Protein Characterization Techniques
- Proteolytic enzymes like trypsin, chymotrypsin, and elastase cleave proteins at specific residues.
- Edman degradation chemically sequences peptides by sequentially removing N-terminal residues.
- Mass spectrometry (MS) identifies proteins and sequences peptides via ionization and mass analysis.
Extinction Coefficient and Protein Quantification
- The extinction coefficient (at 280 nm) depends on tryptophan, tyrosine, and cystine content.
- Knowledge of extinction coefficient allows accurate determination of protein concentration.
X-ray Crystallography
- X-ray crystallography determines 3D structures of proteins by analyzing diffraction patterns from protein crystals.
- Electron density maps are used to model atomic structures of proteins, aiding in functional analysis.
Key Terms & Definitions
- Isoelectric Point (pI) — the pH at which a protein has no net charge and is least soluble.
- Specific Activity — enzyme activity per milligram of total protein; a measure of purity.
- Affinity Chromatography — purification method exploiting specific binding interactions.
- SDS-PAGE — technique separating proteins by size after denaturation with SDS.
- Edman Degradation — stepwise chemical method for sequencing peptide N-termini.
- Mass Spectrometry (MS) — analytical technique to measure mass-to-charge ratio of ions for protein identification.
- Extinction Coefficient — absorbance of a 1 mg/mL protein solution at 280 nm; used in quantification.
- X-ray Crystallography — method for determining atomic structure using X-ray diffraction from crystals.
Action Items / Next Steps
- Review tables and figures of purification schemes and extinction coefficients.
- Visit the ExPASy website to explore protein sequence analysis tools.
- Prepare for lab or assignment on protein purification and characterization methods.