(LECTURE 2) Purification and Assay Methods for Macromolecules of Life Study Guide

PURIFICATION AND ASSAY METHODS FOR STUDYING MACROMOLECULES OF LIFE

• Lecturer: Marta Lesiak, Department of Molecular Biology.

• Institution: Řląski Uniwersytet Medyczny w Katowicach.

• Primary Focus: Methodologies for the disintegration, isolation, purification, and analysis of proteins and nucleic acids (DNA/RNA).

HOMOGENIZATION: DISINTEGRATION OF CELLS AND TISSUES

Homogenization is a mechanical procedure used to release internal macromolecules such as proteins and nucleic acids from the biological environment.

• Methods of Disintegration:

  • Sonication: Use of high-frequency sound waves to disrupt cell membranes.

  • High Pressure: Passing cells through a narrow opening under intense pressure.

  • Mechanical Shaking: Fragmentation of tissue using metal balls and high-frequency shaking.

  • Rotating Pestle (Potter-Elvehjem): Pressuring cells using a tightly fitted rotating Teflon pestle in a glass cylinder.

    • Operational detail: Often performed at $600\,rpm$ while moving the tube slowly up and down as the pestle rotates.

  • Permeabilization: Creating "holes" in the cell membrane using gentle detergents.

• Common Buffer: Tissues are often minced in a $0.25\,M$ sucrose buffer to create a tissue-sucrose homogenate.

ISOLATION OF NUCLEIC ACIDS

• Initial Biological Materials:

  • Peripheral blood, epithelial cells, and cultured skin fibroblasts.

  • Amniotic fluid cells (AFC) and chorion cells.

  • Hair follicle cells, blood spots, and semen spots.

  • Specialized sources: Bone marrow, tissue fragments, skull bone fragments, and tooth fragments.

• DNA Isolation Methods:

  1. Phenol-Chloroform Extraction: A mixture used primarily for the removal of proteins and lipids from the aqueous DNA phase.

  2. Salting Out: Removing proteins from lysed cells using high salt concentrations.

  3. DNA Binding Resin: Utilizing a resin to which DNA binds reversibly, allowing for purification through washing and elution.

• RNA Isolation Methods:

  1. Fractionation: Isolation of specific RNA types from the total cell RNA.

  2. Direct Isolation: Reserved for cells synthesizing a specific RNA in high quantities.

  3. Poly(A) RNA Isolation: Specifically capturing mRNA using the poly-adenylated tail.

  4. Polyribosome Isolation: Capturing RNA directly from the protein-synthesizing machinery.

  5. Total RNA Isolation & RT-PCR: Extracting total RNA followed by the synthesis of complementary DNA ($cDNA$) via Polymerase Chain Reaction.

QUANTITATIVE AND QUALITATIVE ANALYSIS OF NUCLEIC ACIDS

After isolation, purity and concentration must be determined using UV light absorbance or fluorescence.

• UV Light Absorbance Ratios ($A_{260} / A_{280}$):

  • Ratio $1.8 - 2.0$: Indicates pure, double-stranded DNA free from contamination.

  • Ratio > 2.0: Indicates contamination of the DNA sample with RNA.

  • Ratio < 1.0 (approx. $0.5$): Indicates contamination of the DNA sample with proteins.

• Visual Comparison: Comparing ethidium bromide fluorescence of the sample against a control of known concentration.

GEL ELECTROPHORESIS

• Definition: A technique used to separate DNA, RNA, or protein fragments based on their size and charge.

• Mechanism: An electric current is run through a gel (usually agarose or polyacrylamide). Molecules migrate toward the opposite charge.

• Migration speed: Smallest fragments move fastest and furthest; largest fragments move slower and remain closer to the wells.

• Standardization: A "DNA ladder" containing fragments of known sizes is run alongside samples for size estimation.

• Visualizing Tools: Agarose gels for DNA after PCR, RNA gels, and Bioanalyzer chromatograms.

DNA INVESTIGATION METHODS

• Restriction Enzymes (Endonucleases): Enzymes like $EcoRI$ recognize specific sequences (e.g., $GAATTC$) and cut the DNA.

  • Restriction Modification: Enzymes like $EcoRI$ methylase add a methyl group ($CH_{3}$) to the recognition site to prevent cutting by the endonuclease.

• Southern Blotting: A multi-step process to identify specific DNA sequences:

  1. Restriction: DNA is cut by enzymes.

  2. Electrophoresis: Fragments are separated on a gel.

  3. Blotting: DNA is transferred from the gel to a nitrocellulose filter/paper via capillary action using an alkaline solution (which also denatures the DNA).

  4. Hybridization: A radioactive nucleic acid probe binds to its complementary sequence on the filter.

  5. Detection: An autoradiogram is produced by exposing X-ray film to the filter.

• Polymerase Chain Reaction (PCR): Developed in 1983 by Kary Mullis (1993 Nobel Prize).

  • Process: Repeated cycles of heating (Denaturation), cooling (Annealing of primers), and extension (DNA polymerase activity).

  • Ingredients: DNA template, primers, $dNTPs$, and DNA polymerase.

• Sequencing: Determining the exact order of nucleotide bases ($A, T, C, G$).

DNA SEQUENCING (SANGER METHOD)

Developed by Frederick Sanger (1980 Nobel Prize), also known as the dideoxy or chain termination method.

• Required Ingredients:

  • DNA polymerase enzyme.

  • Primer (starter sequence).

  • Four standard DNA nucleotides ($dATP, dTTP, dCTP, dGTP$).

  • Template DNA.

  • Dideoxynucleotides ($ddNTPs$): Each of the four ($ddATP, ddTTP, ddCTP, ddGTP$) is labeled with a different colored fluorescent dye. They lack the $3'-OH$ group required for further polymerization, causing chain termination.

• Analysis: Resulting fragments are separated via capillary gel electrophoresis and read by a laser/detector to produce a chromatogram.

ISOLATION AND PURIFICATION OF PROTEINS

• Source Material: High-abundance sources are preferred. Today, many proteins are produced as recombinant proteins in bacteria, yeast, or mammalian cell cultures.

• Primary Isolation Steps:

  1. fragmentation and crushing (homogenization/ultrasound).

  2. Extraction and removal of debris via centrifugation.

  3. Pre-treatment (optional): Salting out with ammonium sulfate ($(NH_{4})_{2}SO_{4}$).

  4. Purification: Chromatography.

  5. Analysis: Electrophoresis or spectrophotometry.

CENTRIFUGATION TECHNIQUES

• Differential Centrifugation: Successive steps of increasing force to separate organelles:

  • $800\,g$ for $10\,min$: Pellet enriched in nuclei and cellular debris.

  • $20,000\,g$ for $15\,min$: Pellet enriched in mitochondria and chloroplasts.

  • $100,000\,g$ for $60\,min$: Pellet enriched in "microsomes" (plasma and internal membranes).

  • $150,000\,g$ for $3\,hours$: Pellet enriched in ribosomes.

• Density Gradient Centrifugation: Uses a stable sucrose gradient (e.g., $5-20\%$) to separate components by velocity or equilibrium sedimentation.

CHROMATOGRAPHY

Separation based on the affinity of mixture constituents between a mobile phase (fluid) and a stationary phase (material in a column or on a plate).

• Gel-Filtration (Size-Exclusion): Separates proteins by size; larger proteins move around porous beads and elute first, while smaller proteins enter the beads and elute later.

• Ion-Exchange: Proteins are separated based on their net charge. Uses beads with a specific charge (e.g., positive charges to bind negative ions).

• Affinity Chromatography: Uses specific biological interactions. Beads are coated with ligands or antibodies (e.g., anti-tag antibodies) to capture a specific "tagged" protein.

PROTEIN ANALYSIS AND IDENTIFICATION

• SDS-PAGE (Polyacrylamide Gel Electrophoresis):

  • Mechanism: Proteins are denatured and coated with Sodium Dodecyl Sulfate ($SDS$), giving them a negative charge proportional to their length.

  • Reducing Agent: $2-mercaptoethanol$ is used to reduce disulfide bonds ($-S-S- \rightarrow -SH + HS-$).

  • Separation: Proteins move toward the anode based solely on polypeptide chain length.

• Isoelectric Focusing (IEF): Separates proteins based on their isoelectric point ($pI$). Proteins migrate through a pH gradient until they reach a pH where their net charge is zero.

• ELISA (Enzyme-Linked Immunosorbent Assay): Used to quantify proteins using antibodies.

  • Direct ELISA: Antigen-coated well; enzyme-conjugated antibody added.

  • Indirect ELISA: Antigen-coated well; primary antibody added; enzyme-conjugated secondary antibody added.

  • Sandwich ELISA: Antibody-coated well; antigen added; enzyme-conjugated secondary antibody added.

  • Competitive ELISA: Antibody is incubated with the sample antigen before being added to an antigen-coated well.

  • Detection Enzymes: Alkaline phosphatase, Horseradish peroxidase, and Glucose oxidase.

• Protein Identification: Mass Spectrometry ($MS$) analyzes peptide mass fingerprinting. Components include an ion source, mass analyzer, and detector.

STRUCTURAL BIOLOGY METHODS

• X-Ray Crystallography: Determining the 3D position of atoms by analyzing the diffraction pattern of X-rays passing through a protein crystal.

• NMR Spectroscopy (Nuclear Magnetic Resonance): Studies protein structure and conformation in solution.

  • Process: Alignment of nuclear spins in a magnetic field ($B_{0}$), perturbation by radio-frequency (RF) pulses, and analysis of emitted electromagnetic waves.

  • Applications: $^{31}P\,NMR$ can measure shifts in phosphocreatine ($PCr$) and inorganic phosphate ($Pi$) to determine intracellular pH.

• Circular Dichroism (CD): Uses circularly polarized light to determine the folding and thermodynamic stability of proteins.

  • Capabilities: Characterizes secondary structure ($̑$-helix, $̒$-sheet, random coil), thermal stability, and conformational changes upon ligand binding.