Molecular Biology Methods for DNA and Protein Analysis

Posted on Feb 22, 2026 in Biochemistry

Enzyme-Linked Immunosorbent Assay (ELISA)

In an ELISA, an antigen is detected using a primary antibody and a secondary enzyme-linked antibody. The standard procedure involves:

  • Antigen: Immobilization of the target.
  • Wash: Removal of unbound substances.
  • Primary Antibody: Binding to the specific antigen.
  • Wash: Removal of unbound primary antibodies.
  • Secondary Antibody: Binding to the primary antibody.
  • Wash: Removal of unbound secondary antibodies.
  • Substrate Addition: Adding HRP (Horseradish Peroxidase) to react with the enzyme.
  • Signal Measurement: Detecting the resulting color change.

Plasmid Miniprep: DNA Purification

The Plasmid Miniprep process is used to purify plasmid DNA from bacteria. The steps include:

  • Resuspension: Using a solution containing Tris buffer (pH 8.0) for stability, EDTA for protection, Glucose for osmotic balance, and RNAse to degrade RNA.
  • Alkaline Lysis: SDS solubilizes the membrane while NaOH denatures the DNA.
  • Neutralization: A KOAc (pH 5.5) solution allows the plasmid DNA to renature. This is a selective step, as only small, supercoiled plasmid DNA can renature effectively.
  • Centrifugation: The plasmid remains in the supernatant.
  • Binding: Chaotropic agents dissociate water, replacing it with Na+ so that DNA can bind to the column.
  • Washing: Removal of chaotropic agents, EDTA, and extra proteins.
  • Elution: Adding water to detach and collect the pure DNA.

Bacterial Transformation and Competent Cells

Transformation is the introduction of a plasmid into a bacterial cell. The process involves:

  • Transferring colonies to a cold transformation solution containing CaCl2 to disrupt E. coli membranes, creating competent cells.
  • Adding the plasmid.
  • Heat Shock and Recovery: CaCl2 combined with heat shock permeabilizes the E. coli.
  • Adding LB media for growth and pipetting.

Purity is confirmed if the Abs260/Abs280 ratio is >1.8, indicating pure DNA.

Essential Plasmid Elements

  • Origin of replication
  • Control elements for plasmid copy number
  • Genes beneficial to bacteria
  • Antibiotic resistance for selection
  • Foreign gene of interest
  • Recombinant DNA

CRISPR-Cas9 Gene Editing Mechanism

  1. Target: A Guide RNA (gRNA) is designed to match a specific DNA sequence in the target organism’s genome.
  2. Binding: The gRNA binds to the target and recruits the Cas9 enzyme.
  3. Cut: Cas9 creates a double-strand break.
  4. Repair: The cell uses Homology Directed Repair (HDR), often involving Arabinose.

Polymerase Chain Reaction (PCR)

PCR is used for the duplication of DNA. Key components include the template, Taq polymerase, nucleotides, primers, and a Mg+ buffer (a cofactor for Taq that stabilizes duplex formation between the primer and template).

PCR Cycle Steps

  • Denaturation: DNA is heated to 95–98°C to break hydrogen bonds.
  • Annealing: The mixture is cooled to allow primers to bind to the sequence.
  • Extension: The temperature is raised to the optimal level for Taq polymerase to extend the DNA.

Primer Design and Melting Temperature

Primers are typically about 20 nucleotides long. Calculations include:

  • Melting Temperature (Tm): 2 * (A + T) + 4 * (G + C)
  • Annealing Temperature (Ta): Tm – 5

Agarose Gel Electrophoresis

This technique separates DNA, RNA, or proteins based on size and charge. DNA is separated primarily by size. The steps are:

  1. Load samples into the gel.
  2. Apply an electric current.
  3. DNA fragments separate as they migrate.
  4. Stain the gel and visualize under UV light.

Tagged Protein Purification via Affinity

In this method, Histidine tags bind to Ni2+ ions. The process includes:

  • Adding E. coli lysate containing the His-tagged protein.
  • Washing with a low concentration of imidazole to remove impurities.
  • Adding a high concentration of imidazole to elute the purified protein.
  • Collecting and testing for purity.

SDS-PAGE: Protein Separation by Size

SDS-PAGE separates proteins based on size rather than charge. Because proteins have different isoelectric points and charges, SDS (Sodium Dodecyl Sulfate) is used to interact with hydrophobic regions, denaturing them and coating them in a uniform negative charge. This ensures separation is based on linear length rather than 3D structure.

Sample Preparation

Calculate the volume of protein samples to add to the mixture based on the total volume per well and the protein mass. Calculate the required volume of 4X sample buffer and water. Prepare the dilution and heat it in a water bath at 45°C for 10 minutes to denature the proteins.

SDS-PAGE Procedure

  1. Prepare and load the polyacrylamide gel.
  2. Pipette a protein ladder alongside the samples into the wells.
  3. Run the gel at 200V.

SDS-PAGE Reagents and Their Functions

  • Tris: Acts as a pH buffer.
  • SDS: Denatures and coats proteins in a negative charge.
  • Glycerol: Increases sample density so it sinks to the bottom of the well, preventing diffusion.
  • β-Mercaptoethanol: Reduces disulfide bonds to prevent dimer formation.

Gel Composition and Stacking

Since proteins are too small for agarose, polyacrylamide gels are used. The gel consists of two parts:

  • Stacking Gel: Lower acrylamide percentage and lower pH (6.8) to concentrate proteins into narrow bands.
  • Separating (Resolving) Gel: Higher acrylamide percentage and higher pH (8.8).

Gels must be poured between two plates because polymerization is oxygen-sensitive.

Western Blotting for Specific Protein Detection

Western blotting identifies specific proteins within a sample. After SDS-PAGE separation, proteins are transferred onto a membrane. The membrane is incubated with a primary antibody specific to the target protein. A secondary antibody is then added to bind to the primary antibody, amplifying the signal for visualization via staining, fluorescence, or radioactivity.