Gel electrophoresis and SDS-PAGE, and why short peptides are checked by HPLC

Gel electrophoresis separates molecules such as DNA and proteins by pulling them through a gel with an electric current; smaller molecules move further. It is one of the most familiar techniques in biology, used to size DNA fragments and check protein samples. So why don't certificates of analysis for peptides show a gel? This guide explains how gel electrophoresis and SDS-PAGE work, what they can and can't show, and why short peptides are tested by HPLC and mass spectrometry instead.

Terms are defined in the glossary.

What is gel electrophoresis?

"Electrophoresis" means movement in an electric field. Molecules that carry a charge are drawn towards the electrode of opposite charge: negatively charged molecules move towards the positive electrode (the anode), and positive ones towards the negative electrode (the cathode).

In gel electrophoresis, the molecules have to travel through a gel, a mesh of long polymer chains soaked in buffer. The mesh slows large molecules more than small ones. After a set time, molecules of different sizes have travelled different distances, and each kind forms a band.

How far a molecule moves depends on:

• its charge: more charge means a stronger pull,
• its size and shape: bigger or bulkier molecules are slowed more by the gel,
• the gel's pore size: a denser gel separates smaller molecules, and
• the voltage and run time.

Agarose and polyacrylamide

PAGE stands for polyacrylamide gel electrophoresis.

Gel electrophoresis of DNA

DNA is the most common thing run on a gel, in school labs as well as research. Its phosphate backbone carries a negative charge along its whole length, so every fragment has about the same charge for its size and moves towards the positive electrode, sorted by size alone (Lee et al., J. Vis. Exp. 2012).

• The gel. Agarose gels of 0.5% to 2% suit most fragments, with stronger gels for smaller pieces. Standard agarose gels work best from about 100 base pairs to 25,000; smaller fragments are better separated on polyacrylamide (Lee et al.).
• The DNA ladder. A mixture of DNA fragments of known sizes is run in its own lane. The distance a fragment travels falls with the logarithm of its size, so plotting the ladder gives a scale for reading off the size of each sample band (Lee et al.).
• The stain. DNA is invisible until stained. Ethidium bromide slots between the DNA bases and glows orange under ultraviolet light; SYBR dyes are more sensitive alternatives (Lee et al.).

SDS-PAGE

Proteins are harder to separate by size than DNA, because each protein has its own charge and shape. SDS-PAGE solves this.

1. Denature with SDS. Sodium dodecyl sulfate (SDS) is a detergent with a negative charge. It unfolds proteins and coats them, binding at roughly 1.4 g of SDS per gram of protein. Every protein ends up as a negatively charged rod with a similar charge for its length.
2. Break disulfide bonds. A reducing agent such as dithiothreitol (DTT) or β-mercaptoethanol breaks the disulfide bonds that hold chains together, so each chain runs on its own.
3. Heat the sample briefly to finish unfolding it.
4. Load and run. Samples go into wells at the top of the gel, alongside a molecular weight marker, a ladder of proteins of known size. Current is applied and the proteins move down towards the anode.
5. Stain. The gel is soaked in a dye that binds protein, so the bands show up.

Because SDS has evened out charge and shape, the distance travelled depends mainly on size. Comparing a band with the marker ladder gives its approximate molecular weight. This method, with a dense resolving gel under a looser stacking gel that concentrates the sample into a sharp line first, is the one Laemmli described (Laemmli, Nature 1970).

Staining

What SDS-PAGE is good for

• Checking a protein sample's size and whether it is mostly one protein.
• Seeing whether a protein has been cut or has formed larger aggregates.
• Following a protein through purification.
• As the first step of a western blot, where proteins are transferred from the gel to a membrane and detected with antibodies.

Isoelectric focusing and 2D gels

Isoelectric focusing (IEF) separates molecules by charge rather than size. The gel contains a pH gradient. Each molecule moves until it reaches the pH where its net charge is zero, its isoelectric point, and stops there.

Running IEF in one direction and SDS-PAGE across it gives a two-dimensional (2D) gel, which spreads thousands of proteins into separate spots by charge and size.

Capillary electrophoresis

Capillary electrophoresis (CE) runs the same principle in a very thin glass tube instead of a slab of gel, with a detector at the far end. It is automated and quantitative, and gives a trace with peaks much like a chromatogram. CE can separate small peptides and is sometimes used alongside HPLC as an orthogonal method: a second technique that separates by a different property, to catch impurities the first might miss.

Why short peptides aren't checked on gels

Most synthetic research peptides are between a few hundred and a few thousand daltons. That is 0.3 to 5 kDa, well below the useful range of a standard SDS-PAGE gel. Several things go wrong at that size:

• They don't separate. Below about 10 kDa, standard gels can't tell molecules apart. Small peptides run together at the bottom of the gel with the dye front, or out of it entirely.
• They don't stay put. Short peptides are not held well by the gel and can wash out during fixing and staining.
• They stain poorly. Dyes like Coomassie bind fewer sites on a short chain, so a small peptide gives a weak band or none.
• SDS doesn't even out their charge. Short peptides bind SDS less predictably, so their position doesn't reliably reflect their size.
• The differences that matter are tiny. A typical impurity in a synthetic peptide is a sequence with one amino acid missing or one atom changed, such as an oxidised or deamidated form. That can be a difference of 1 Da in 1,400. No gel can resolve that.

Tricine-SDS-PAGE, a variation with a different buffer system, extends the useful range down to about 1–5 kDa (Schägger, Nat. Protoc. 2006). It can show a peptide's approximate size, but not its purity to a fraction of a percent.

What's used instead

For larger proteins, SDS-PAGE remains a quick and useful check. For short peptides, a gel can't answer the questions a certificate of analysis needs to answer.

Related guides

For how chromatography separates molecules, see how chromatography works, and for the paper and thin layer versions, where spots are measured by how far they travel, see Rf values and thin layer chromatography. For reading the trace that HPLC produces, see reading a chromatogram. For how peptides are made and why their impurities look the way they do, see solid-phase peptide synthesis. An independent lab tests every one of our lots for purity by HPLC and identity by mass spectrometry. Every lot has its own COA, published on the product page and on that lot's own page. See Quality.