Gel Electrophoresis: Separating DNA and Proteins

Chromatography separates molecules by how they distribute between two phases. Electrophoresis separates them in a different way: by pulling charged molecules through a gel with an electric field. It’s the workhorse technique of molecular biology and biochemistry, used to compare DNA in forensic cases, diagnose genetic conditions, check the purity of proteins and verify every step of genetic engineering.

The basic principle

Charged particles in an electric field move towards the electrode of opposite charge:

• negatively charged molecules (anions) move towards the positive electrode (anode)
• positively charged molecules (cations) move towards the negative electrode (cathode)

How fast a molecule moves depends on:

• its charge: more charge means a bigger pulling force
• its size and shape: larger molecules are held back more by the medium
• the strength of the field (voltage)
• the medium it moves through

In gel electrophoresis, the medium is a gel: a network of cross-linked polymer chains full of tiny pores, soaked in a buffer solution. The gel acts like a molecular sieve: small molecules slip through the pores easily and travel far; large molecules get tangled and move slowly.

Why DNA separates by size

DNA has a phosphate group in every nucleotide of its backbone. At the pH of the running buffer (around 8), each phosphate is ionised and carries a negative charge. So:

• every DNA fragment is negatively charged and moves towards the positive electrode
• the charge is proportional to length (one negative charge per nucleotide), so the charge-to-mass ratio is roughly the same for all DNA fragments

Because the pulling force per unit mass is about equal for all fragments, the only thing that differs is how much the gel slows them down, which depends on size. Short fragments travel furthest; long fragments stay near the start.

This is why DNA fragments are always loaded at the negative end of the gel. A useful memory aid: “run to red”, since the positive lead is usually red.

Running a DNA gel: step by step

1. Make the gel. Agarose powder (a polysaccharide purified from seaweed) is dissolved in hot buffer, poured into a tray with a comb to form wells, and left to set.
2. Place the gel in a tank and cover it with buffer, which conducts electricity and keeps the pH stable. See buffers explained.
3. Prepare samples. DNA is mixed with a dense loading dye, which contains glycerol (so the sample sinks into the well) and a coloured tracking dye (so you can see how far the run has progressed).
4. Load the wells with a micropipette, including a DNA ladder: a mixture of fragments of known sizes.
5. Connect the power supply, with the wells at the negative electrode, and run at a set voltage (often around 100 V for a small gel) for 30–60 minutes.
6. Stain and visualise. DNA is colourless, so it’s stained with a dye that binds to DNA and fluoresces under UV or blue light, showing each group of same-sized fragments as a band.

Reading the gel

The DNA ladder acts like a ruler. Fragment sizes are measured in base pairs (bp). By comparing the distance travelled by an unknown band with the ladder bands, its size can be estimated. Over the working range, distance travelled is roughly proportional to the logarithm of fragment size, so a plot of log(size) against distance gives a near-straight calibration line.

Example: A ladder shows bands at 1000, 500 and 250 bp. An unknown band lies about halfway between the 1000 and 500 bp bands. Because the relationship is logarithmic, its size is about √(1000 × 500) ≈ 700 bp, not 750.

Agarose vs polyacrylamide gels

	Agarose	Polyacrylamide (PAGE)
Source	natural polysaccharide from seaweed	synthetic polymer made from acrylamide
Pore size	larger	smaller, and easily controlled
Best for	DNA fragments from about 100 bp to tens of thousands of bp	proteins; small DNA fragments differing by a single base
Preparation	melt and pour; easy and safe	polymerised chemically; acrylamide monomer is toxic before polymerisation

Changing the gel concentration changes the pore size: a higher percentage gel has smaller pores and separates small fragments better.

Proteins: SDS-PAGE

Proteins are harder to separate by size, because their charges vary (depending on their amino acids and the pH, see amino acids as acids and bases) and their shapes vary too.

SDS-PAGE solves this:

• The protein is heated with SDS (sodium dodecyl sulfate), a detergent, often with a reducing agent that breaks disulfide bridges.
• SDS unfolds the protein into a roughly linear chain and coats it with negative charges, approximately in proportion to its length.
• Every protein now has a similar charge-to-mass ratio and a similar shape, so, just like DNA, it separates by size in a polyacrylamide gel.

Protein sizes are measured in kilodaltons (kDa) against a protein ladder, and bands are stained with dyes such as Coomassie blue.

Other types of electrophoresis

• Native PAGE: proteins run without SDS, keeping their natural shape and charge, so the separation depends on both.
• Isoelectric focusing: proteins move through a pH gradient until they reach their isoelectric point, the pH at which their net charge is zero, and stop there.
• 2D electrophoresis: isoelectric focusing in one direction, then SDS-PAGE at right angles, separating thousands of proteins on one gel.
• Capillary electrophoresis: separation in a very thin capillary tube, with automated detection. It’s used in modern DNA sequencing and forensic DNA profiling, and for analysing small ions and drugs.

Uses

• Forensic DNA profiling: comparing lengths of repeated DNA sequences (short tandem repeats) between a crime-scene sample and a suspect, now usually by capillary electrophoresis. See forensic chemistry.
• Paternity and relationship testing: the same principle.
• Medical diagnosis: detecting genetic mutations; serum protein electrophoresis to spot abnormal proteins in blood; distinguishing normal and sickle-cell haemoglobin.
• Genetic engineering: checking that DNA has been cut or copied correctly (for example, after the polymerase chain reaction, PCR).
• Protein research: checking purity and estimating molecular mass.

Safety

• Electrophoresis tanks use high voltages; never open the lid or touch the buffer while the power is on.
• UV light damages eyes and skin; use a shield or safer blue-light systems.
• Some DNA stains are hazardous; follow the safety data sheet and wear gloves. See how to read a safety data sheet.
• Unpolymerised acrylamide is a neurotoxin; pre-made gels reduce exposure.

Key takeaways

• Electrophoresis separates charged molecules in an electric field through a gel that acts as a sieve.
• DNA is negatively charged (phosphate backbone) and moves towards the positive electrode; smaller fragments travel further.
• A DNA ladder of known sizes is used to estimate unknown fragment sizes, using a logarithmic relationship.
• Agarose gels suit DNA; polyacrylamide gels suit proteins and small DNA fragments.
• SDS-PAGE gives proteins a uniform charge and shape so they separate by size.