Biochemistry - Lesson 3: Protein Purification and Analysis Techniques
Biochemical research often relies on isolating and analyzing specific proteins to study their functions, structures, and interactions. This lesson provides an in-depth look at common laboratory methods for protein purification and characterization, emphasizing how each technique exploits distinct physicochemical properties of proteins.
Protein Extraction and Fractionation
The first step is to obtain a crude extract rich in the target protein. Cells are disrupted by cell lysis (using mechanical methods, detergents, or sonication), releasing intracellular contents. Subsequent fractionation and centrifugation steps help separate soluble proteins from insoluble material:
- Low-speed centrifugation: Removes cell debris and unbroken cells.
- High-speed centrifugation: Pellets organelles; supernatant contains cytoplasmic proteins.
- Ultracentrifugation: Can further separate components by mass or density (e.g., ribosomal fractions).
This clarified lysate serves as the starting point for more specific purification techniques.
Chromatography Techniques
Chromatography separates proteins based on properties such as charge, size, affinity, or hydrophobicity. A chosen method often depends on the protein’s known attributes:
| Technique | Principle | Common Usage |
|---|---|---|
| Ion-Exchange Chromatography | Exploits net charge differences; proteins bind to oppositely charged resin | Separating acidic/basic proteins; adjusting salt or pH elutes bound proteins |
| Gel Filtration (Size-Exclusion) | Separates by size; larger proteins elute first, bypassing pores in beads | Estimating molecular weight or removing small contaminants |
| Affinity Chromatography | Uses specific binding interactions (antibody, substrate analog, metal chelate) | High specificity purifications (e.g., His-tag on Ni2+ resin) |
| Hydrophobic Interaction | Exploits hydrophobic patches; high salt promotes binding, low salt elutes | Purifying nonpolar proteins or capturing hydrophobic domains |
Multiple chromatographic steps are often combined for maximum purity. Early steps (like ion-exchange) remove bulk contaminants; later steps (affinity) achieve higher specificity.
The chart above illustrates a typical trade-off: as purification steps progress, yield decreases, but purity increases. Each method narrows in on the protein of interest while discarding other components.
Electrophoretic Methods
Once a partially or fully purified protein sample is obtained, electrophoresis helps assess purity, subunit composition, and size:
- SDS-PAGE (Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis) Proteins are denatured with SDS and migrate primarily according to molecular weight. Smaller proteins run faster through the gel. A molecular weight marker can estimate protein size.
- Isoelectric Focusing Proteins migrate in a pH gradient gel until reaching their isoelectric point (pI). Useful for separating proteins with subtle charge differences.
In advanced workflows, 2D electrophoresis combines isoelectric focusing (1D) and SDS-PAGE (2D) for higher resolution separation, mapping complex proteomes.
Protein Sequencing and Structure Determination
Characterizing primary structure or higher-order structure often follows purification. Key approaches include:
- Edman Degradation Sequentially labels and cleaves N-terminal residues to determine amino acid sequence. Primarily for smaller peptides.
- Mass Spectrometry Measures mass-to-charge ratios of peptide fragments. Modern methods (ESI, MALDI-TOF) rapidly determine large protein sequences and post-translational modifications.
- X-ray Crystallography Requires crystallizing the protein; provides high-resolution atomic coordinates. Long a gold standard for 3D structure.
- NMR Spectroscopy Exploits magnetic properties of atomic nuclei. Offers 3D structures in solution, valuable for proteins < ~30 kDa.
These techniques reveal how amino acid sequence and spatial conformation dictate protein activity, stability, and interactions. A combination of methods (e.g., mass spectrometry for sequence, X-ray or cryo-EM for structure) often yields a comprehensive molecular profile.
Practical Considerations
Buffer selection and pH control are critical throughout purification to maintain protein stability. Protease inhibitors often prevent protein degradation in cell extracts, while temperature control (e.g., working on ice) preserves delicate complexes. The final goal is to retain the native conformation for functional studies or to obtain a homogeneous sample for structural analysis.
Summary
Effective protein purification underpins biochemical analysis, requiring strategic use of fractionation, chromatography, and electrophoretic techniques. Subsequent sequencing and structural determination methods illuminate how protein properties correlate with biological function. This integrated approach—combining purification, characterization, and structural elucidation—forms the basis of modern biochemical research.
Suggested Reading:
Lehninger Principles of Biochemistry (chapters on protein purification and characterization)
Biochemistry by Berg, Tymoczko, and Stryer (sections covering chromatographic separation, SDS-PAGE, and structure determination)
