6.1.3(e) - Electrophoresis for nucleic acids and proteins

Electric Field And Gel

Electrophoresis means movement of charged particles in an electric field. In gel electrophoresis, the particles move through a gel matrix. The gel contains pores, so it acts like a molecular sieve: smaller molecules or fragments pass through the pores more easily than larger ones.

The gel sits in a buffer solution. The buffer conducts current and helps keep the pH stable. Samples are loaded into small wells at one end of the gel, then a potential difference is applied across the gel.

The electrodes matter. Negatively charged molecules move towards the positive electrode, called the anode. Positively charged molecules move towards the negative electrode, called the cathode. Nucleic acids such as DNA and RNA are negatively charged because of phosphate groups in their sugar-phosphate backbones, so they move towards the anode.

For nucleic acid fragments of the same type, shorter fragments usually travel further through the gel in a fixed time. A longer DNA fragment has more negative charges, but it also has more mass and is slowed more by the gel pores. The separation is therefore mainly by fragment length, with shorter fragments moving further from the wells.

Proteins are more complicated because their amino acid sequences give them different shapes and different net charges. A protein's movement may depend on its net charge, size and shape unless the method deliberately reduces the effect of shape and charge.

[DIAGRAM: gel-electrophoresis-migration: Lesson 152: Electrophoresis For Nucleic Acids And Proteins - diagram 01; asset_slug: 152_m06_1_3_electrophoresis_for_nucleic_acids_and_proteins__diagram_01; recommended_method: drawn_biology; description: A 16:9 drawn gel electrophoresis setup with wells near the cathode, anode at the far end, buffer, gel pores, and three negatively charged DNA fragments showing smaller fragments migrating further through the gel.]

Nucleic Acid Fragments

When electrophoresis is used for nucleic acids, the sample may contain DNA or RNA fragments of different lengths. A mixture might come from a PCR reaction, a restriction-enzyme digest, a DNA profiling investigation, or another molecular analysis. Those neighbouring techniques are not the focus here; the point is that electrophoresis separates the fragments so the pattern can be analysed.

The practical logic is simple:

A lane is one track through the gel from one well. A band is a visible group of many fragments that have travelled to the same position. If a sample produces two bands, it contains at least two groups of fragments with different lengths, or at least two groups that migrated differently under those conditions.

When a band in an unknown lane is at the same position as a marker band, it suggests that the unknown fragment has a similar length to that marker fragment. It does not prove that the unknown has the same base sequence. Different fragments can have the same length.

Protein Electrophoresis

Electrophoresis can also separate proteins. This is useful because cells often contain mixtures of many proteins, and a researcher may want to compare protein samples, estimate the relative molecular mass of a protein, or check whether a purification step has removed other proteins.

The same general idea applies: molecules move in an electric field and the gel slows them. The difference is that proteins do not all have the same charge pattern. Their side chains can be positive, negative, polar or non-polar, and the folded shape of a protein can affect how easily it passes through the gel.

In native protein electrophoresis, proteins keep more of their natural structure. Their mobility can depend on net charge, size and shape. That can be useful for some comparisons, but it makes simple size interpretation harder.

A common protein method is SDS-PAGE. SDS is a detergent that denatures many proteins and gives them a more uniform negative charge relative to their mass. In that method, the proteins move towards the positive electrode and separate mainly by molecular mass. Smaller polypeptides usually move further through the gel than larger polypeptides.