Makindo Medical Notes

Related Subjects: |Major Histocompatibility complex |Blotting Techniques: Gel Electrophoresis

Blotting is a core technique in molecular biology used to detect specific macromolecules (DNA, RNA, or proteins) in a sample. It typically follows gel electrophoresis, which separates molecules based on size and charge, allowing for individual fragment analysis.

Introduction to Gel Electrophoresis

Gel Electrophoresis separates macromolecules (DNA/RNA, proteins) based on size and charge:

• Size and Mobility: Larger molecules move more slowly through the gel matrix, while smaller molecules travel further.
• Charge: Molecules with less negative charge move more slowly toward the positive electrode.
• Secondary/Tertiary Structures: Structural folding can affect movement. Denaturing agents, such as SDS for proteins or urea for nucleic acids, are used to disrupt these structures so that molecular size primarily determines mobility.

Blotting Techniques Overview

Different blotting methods detect specific macromolecules, each using unique approaches and probes to identify target sequences or structures:

Detailed Descriptions of Blotting Techniques

Southern Blotting

Named after Dr. Edwin Southern, this technique detects specific DNA sequences.

• Preparation: Double-stranded DNA is cut with restriction enzymes to produce smaller fragments.
• Gel Electrophoresis: Fragments are separated by size, creating a smear on the gel.
• Blotting: DNA is transferred to a nylon or nitrocellulose membrane via capillary action.
• Hybridization: Labelled DNA probes complementary to the target sequence are added. Probes may be labelled with radioisotopes (e.g., ³²P), fluorescent tags, or enzymes for detection.
• Detection: The membrane is washed to remove non-specific binding, and probes are detected using autoradiography, chemiluminescence, or fluorescence.

Northern Blotting

Similar to Southern blotting but used for RNA detection, allowing for the study of gene expression.

Western Blotting

Used to detect specific proteins, with wide applications in research and diagnostics, such as detecting protein mutations in diseases:

• Protein Separation: Proteins are separated by SDS-PAGE, a form of gel electrophoresis for proteins.
• Blotting: Separated proteins are transferred to a membrane (e.g., PVDF or nitrocellulose).
• Probing: Antibodies specific to the protein of interest are added. Antibodies are often linked to enzymes or fluorescent tags for visualization.
• Applications: Western blotting is used in studies of protein modifications, mutations, and quantification. For instance, sickle cell disease involves a mutation in hemoglobin detectable by Western blot.

Additional Notes

Blotting techniques are vital tools in molecular biology and medicine, enabling detailed analysis of genetic information, gene expression, and protein structure. They are integral in diagnostics, research, and the study of disease mechanisms.