Gel Electrophoresis

How the gel sieves molecules

Pour molten agarose or polymerize acrylamide into a tray with a comb. As it cools, the polymer chains form a 3D mesh whose pore size depends on the gel concentration. Slip the comb out, leaving wells. Submerge the gel in a buffer that conducts current. Load samples into the wells with a tracking dye that lets you see the front. Hook electrodes to a power supply, switch on, and watch.

The samples carry charge: DNA's phosphate backbone is uniformly negative; proteins coated with SDS are uniformly negative. The electric field pushes them toward the positive electrode. To get there, they have to thread through the gel's pores. A 100 base-pair fragment slips through quickly; a 10,000 bp fragment crawls. After 30–90 minutes, the molecules separate into bands at different positions down the lane.

The relationship between distance migrated and molecular weight is log-linear in the gel's resolving range:

distance ∝ log(1 / MW)

so:  log(MW_unknown) = m · d_unknown + b

where m, b come from the ladder calibration.

The setup

            ┌────────────────────────────────┐
            │   power supply  100–200 V DC   │
            └──┬──────────────────────────┬──┘
               │ (−)                  (+) │
               ▼                          ▼
       ┌────────────────────────────────────┐
       │   ╔════════════════════════════╗   │
       │   ║ buffer (TAE / TBE / Tris)  ║   │
       │   ║                            ║   │
       │   ║  ▢▢▢▢▢▢▢▢▢▢  ← wells       ║   │
       │   ║  │ │ │ │ │ │                ║   │
       │   ║  ▼ ▼ ▼ ▼ ▼ ▼  migration    ║   │
       │   ║                            ║   │
       │   ║  ─── ─── ─── ─── ─── ───  ║   │  bands
       │   ║                            ║   │
       │   ║  agarose / polyacrylamide  ║   │
       │   ╚════════════════════════════╝   │
       └────────────────────────────────────┘
                       cassette / tank

For DNA, the gel sits horizontally submerged in buffer. For proteins, the gel runs vertically between two glass plates with buffer above and below — the geometry forces a sharper field and gives finer resolution at smaller scale.

Worked example — reading a protein from a ladder

You ran SDS-PAGE on a recombinant protein next to a Precision Plus ladder. Measured migration distances from the well:

Ladder band (kDa)	Distance (mm)	log(kDa)
250	5.0	2.398
150	9.0	2.176
100	13.5	2.000
75	17.0	1.875
50	23.0	1.699
37	27.5	1.568
25	34.0	1.398
Unknown	20.5	?

Plot log(kDa) on the y-axis and distance on the x-axis. A linear fit through the ladder gives roughly slope m = −0.034, intercept b = 2.57. For the unknown at d = 20.5 mm:

log(MW) = −0.034 × 20.5 + 2.57 = 1.873
MW ≈ 10^1.873 ≈ 75 kDa

Compare to the expected 73 kDa from the gene sequence. The 2 kDa discrepancy is well within the ±5–10% accuracy of SDS-PAGE for a normal globular protein. If your unknown lands far from the prediction, suspect post-translational modification (glycosylation adds 5–30 kDa), proteolytic cleavage, or that you've expressed the wrong fragment.

Six electrophoresis modes compared

Mode	Gel matrix	Separates by	Resolution	Detection	Best for
Agarose (DNA)	0.7–4% agarose	Size (50 bp – 50 kb)	~10% length	Ethidium bromide / SYBR + UV	PCR products, restriction digests
SDS-PAGE	6–20% polyacrylamide	Molecular weight	~5–10% MW	Coomassie / silver / Western	Protein sizing, purity check
Native PAGE	4–15% polyacrylamide, no SDS	Size + charge + shape	Variable	Coomassie / activity stain	Enzyme complexes, oligomers
Isoelectric focusing (IEF)	pH gradient gel	Isoelectric point pI	0.01 pH unit	Coomassie / silver	Charge variants, hemoglobin
2D electrophoresis	IEF strip + SDS-PAGE	pI then MW	~10⁴ spots resolvable	Silver / fluorescent	Whole proteome maps
Capillary electrophoresis	Buffer-filled capillary	Charge / mass / size	10⁵+ theoretical plates	UV / fluorescence / MS	Sanger sequencing, glycans
Pulsed-field gel (PFGE)	1% agarose, alternating field	Very large DNA (50 kb – 10 Mb)	~10%	Ethidium bromide	Bacterial chromosomes, yeast

Sanger DNA sequencing — the technique that finished the Human Genome Project's reference assembly — is fundamentally capillary electrophoresis with single-base resolution and four-color fluorescent detection.

Visualizing the bands

• Ethidium bromide — intercalates DNA and fluoresces orange under UV. Detection limit ~1 ng. Mutagenic; many labs have switched to SYBR Safe or GelRed.
• Coomassie Brilliant Blue — protein dye, ~50 ng detection limit, blue staining requires destaining in acetic acid.
• Silver staining — much more sensitive, ~1 ng of protein, but non-quantitative and finicky.
• Western blot — transfer the gel to nitrocellulose, probe with antibody, detect by chemiluminescence. Specific to one target protein, 1–10 pg sensitivity.
• Stains-all / fluorescent dyes — modern alternatives like SYPRO Ruby give protein detection sensitivity rivaling silver but with linear quantification across three orders of magnitude.

Variants and adjacent techniques

• Pulsed-field gel electrophoresis (PFGE) — alternates the field direction every few seconds; large DNA reorients each switch, separating up to 10 Mb. Used for bacterial epidemiology fingerprinting.
• Two-dimensional electrophoresis (2DE) — first dimension is isoelectric focusing, second is SDS-PAGE perpendicular. Spreads thousands of proteins across the page.
• Northern blot / Southern blot — gel + transfer + probe for RNA and DNA respectively. Largely replaced by qPCR and sequencing but still used for transcript size confirmation.
• Microfluidic chip electrophoresis (Bioanalyzer, TapeStation) — miniaturized capillary system that runs a sample in 30 seconds with built-in sizing.

Common pitfalls

• Smile-shaped lanes. The gel got too hot and the edges ran faster. Lower voltage, run at 4 °C, or use a buffer cooler.
• Bands fall off the bottom. Watch the dye front and stop the run before it reaches the gel edge — the gradient is gone past that point.
• No bands at all. Check polarity (DNA migrates to the +). Most power-supply mistakes are reversed leads.
• Protein streaks vertically. Inadequate denaturation — boil samples in 2× Laemmli at 95 °C for 5 minutes with fresh DTT or β-mercaptoethanol.
• Salt fronts. High ionic strength in the sample distorts the leading edge. Desalt the protein, or add SDS sample buffer 1:1 to dilute.
• Old buffer. TAE pH drifts after multiple runs; the current rises and bands smear. Toss after 3–4 runs.

Why electrophoresis still matters

Despite mass spectrometry and high-throughput sequencing taking over identification work, gels remain the cheapest, fastest way to ask "is my purification clean?" or "did my PCR work?". A 1% agarose gel costs about $0.50 in reagents and tells a graduate student in 45 minutes what would otherwise need a $400 sequencing reaction. Every molecular biology lab on Earth runs at least one gel a day — and the apparatus has barely changed since Oliver Smithies invented starch-gel electrophoresis in 1955.