Rucete ✏ Lehninger Principles of Biochemistry In a Nutshell
3.3 Working with Proteins
This chapter introduces the core laboratory techniques used to isolate, purify, separate, and analyze proteins, and explains how researchers detect and quantify proteins during the purification process.
Protein Separation and Purification
• Proteins must be purified from complex mixtures using their distinct physical and chemical properties.
• Initial steps include breaking open cells to obtain a crude extract and, if necessary, fractionating cell components using centrifugation.
• Fractionation methods exploit protein solubility differences using pH, temperature, and salt concentration changes ("salting out" with ammonium sulfate is common).
• Dialysis uses a semipermeable membrane to remove small solutes while retaining proteins.
• Column chromatography is the most effective purification technique and separates proteins based on charge (ion exchange), size (size-exclusion/gel filtration), and binding affinity (affinity chromatography).
• High-performance liquid chromatography (HPLC) provides rapid, high-resolution separation using high pressure and improved materials.
• Protein purification usually involves multiple sequential methods to achieve high purity, as documented in a purification table tracking yield and specific activity.
Column Chromatography Methods
• Ion-exchange chromatography separates proteins based on their net charge at a given pH; cation exchangers bind positively charged proteins, anion exchangers bind negatively charged proteins.
• Size-exclusion chromatography separates by size; larger proteins elute faster because they bypass the pores of the stationary phase.
• Affinity chromatography uses columns with covalently attached ligands that bind only specific proteins, enabling one-step purification of tagged or naturally binding proteins.
• Genetically engineered tags can be added to proteins for simplified affinity purification, with tags often removable after isolation.
Electrophoresis and Analytical Methods
• Electrophoresis in polyacrylamide gels separates proteins based on charge-to-mass ratio and size, providing information on purity and molecular weight.
• SDS-PAGE (with sodium dodecyl sulfate) denatures proteins and gives them uniform negative charge, so separation is based almost entirely on size; smaller proteins migrate faster.
• Proteins are visualized using dyes like Coomassie blue; comparison with molecular weight standards allows for mass estimation.
• Isoelectric focusing separates proteins based on their isoelectric point (pI) by establishing a pH gradient in the gel; proteins migrate until reaching the pH matching their pI.
• Two-dimensional electrophoresis combines isoelectric focusing and SDS-PAGE, resolving thousands of proteins by both pI and molecular weight.
Detection and Quantification of Proteins
• Assays are needed to detect and measure the protein of interest at every step; enzymes are typically assayed by their catalytic activity under defined conditions.
• Activity refers to the total amount of functional protein (e.g., enzyme units); specific activity is the activity per milligram of total protein, indicating purity.
• Specific activity increases as purification progresses and reaches a maximum when the protein is pure.
• Non-enzymatic proteins can be assayed by binding assays, biological effects, or abundance in tissue extracts.
In a Nutshell
• Protein purification relies on techniques that separate proteins by solubility, size, charge, and affinity, often using multiple chromatography methods sequentially.
• Analytical tools like electrophoresis and specific assays are critical for monitoring purity, determining molecular weight, and identifying proteins during purification.
• Mastery of these methods is fundamental for studying protein structure, function, and for advancing research in biochemistry and molecular biology.