Undergraduate · 20 min

Paper electrophoresis: separating proteins by charge

Spot three model proteins on a buffer-soaked strip, switch on the field, and watch them part — or reverse direction — as the pH changes.

Goal

Predict the sign of each protein's net charge from $pI$ vs $pH$ , rank the bands by migration, and see why the buffer pH is the master switch.

Apparatus and reagents

Virtual bench: paper strip, buffer reservoirs, DC supply, staining tray; proteins A (pI 4.5), B (pI 6.7) and C (pI 9.5).

Procedure

Stage 0 shows the sample spotted at the origin. Switch to stage 1 (pH 8.6): which band moves fastest, and why?
Check that at pH 8.6 all bands travel toward the anode (+). Rank A, B, C by net charge using the labels.
Switch to stage 2 (pH 6.0). Which protein reverses direction, and which one barely moves?
For each protein, compare pH with its pI and state whether it is a cation, nearly neutral, or an anion at each stage.

What to observe

At pH 8.6 all three proteins carry a net negative charge, so all migrate toward the anode; protein A, farthest from its pI, travels farthest.
At pH 6.0, B sits near its pI and stays almost put, while C (pI 9.5 > pH) is protonated and migrates to the cathode.

Explanation

A protein is a polyelectrolyte: below its isoelectric point $pI$ it is protonated and positive, above it deprotonated and negative, and at $pH = pI$ its net charge is zero. In a field $E$ the drift speed is set by the net charge divided by the frictional drag of the paper and solvent, so $v \propto q_\mathrm{net}E$ . Two runs at different pH values can therefore sort the same mixture both by how fast bands move and by which way they go.

History of the experiment

Arne Tiselius built the first moving-boundary electrophoresis apparatus in Uppsala in the 1930s, winning the 1948 Nobel Prize in Chemistry. Paper-based versions spread through hospital labs in the 1940s–50s to profile serum proteins, before gels and capillaries took over.

Chemists behind it

Frederick Sanger