Understanding:
• Gel electrophoresis is used to separate proteins or fragments of DNA according to size
Gel electrophoresis is a laboratory technique used to separate and isolate proteins or DNA fragments based on mass / size
- Samples are placed in a block of gel and an electric current is applied which causes the samples to move through the gel
- Smaller samples are less impeded by the gel matrix and hence will move faster through the gel
- This causes samples of different sizes to separate as they travel at different speeds
Overview of Gel Electrophoresis
While both DNA and proteins are separated according to the same basic process, differences exist between the two protocols
DNA Separation
- DNA may be cut into fragments using restriction endonuclease – different DNA samples will generate different fragment lengths
- Fragments separate because DNA is negatively charged due to the presence of a phosphate group (PO43–) on each nucleotide
- DNA samples are placed into an agarose gel and fragment size calculated by comparing against known industry standards
- Specific sequences can be identified by incorporating a complementary radiolabelled hybridisation probe, transferring the separated sequences to a membrane and then visualising via autoradiography (Southern blotting)
Agarose Gel Electrophoresis (DNA)
Protein Separation
- Proteins may be folded into a variety of shapes (affecting size) and have positive and negative regions (no clear charge)
- Proteins must first be treated with an anionic detergent (SDS) in order to linearise and impart a uniform negative charge
- Protein samples are placed into a polyacrylamide gel and sizes compared against known industry standards
- Separated proteins are transferred to a membrane and then target proteins are identified by staining with specific monoclonal antibodies (Western blotting)
Polyacrylamide Gel Electrophoresis (Proteins)
