2D gel electrophoresis
Separate proteins by isoelectric point, then by size.
Our ruleSimulations prepare you for the bench but don't replace hands-on practice.
1Two separations that share nothing
A one-dimensional gel can only separate proteins by one property, and a cell makes far too many proteins for any single property to tell them apart. Two-dimensional electrophoresis uses charge and then size, which is a good pairing because the two are nearly independent: knowing a protein's isoelectric point tells you almost nothing about its mass.
2What only a 2D gel shows: proteoforms
Most post-translational modifications change a protein's charge without changing its mass appreciably. A phosphate adds about 80 Da to a 40,000 Da protein, invisible on SDS-PAGE, but takes roughly 0.05–0.1 units off the isoelectric point.
Clean up and rehydrate
Precipitate or desalt the sample, digest the nucleic acid, and rehydrate the strip in urea, thiourea, a zwitterionic detergent and carrier ampholytes.
Focus
Ramp the voltage as the conductivity falls, ending at several thousand volts. Each protein stops at its pI.
Equilibrate
Reduce the strip with DTT, then alkylate with iodoacetamide so the thiols cannot re-oxidise in the second dimension.
Run and image
Lay the strip on an SDS gel, run, stain and image. Spot detection, warping and matching are done in software.
3Why the first dimension fails
Isoelectric focusing is a conductivity experiment as much as a separation. Current flows through whatever carries charge, and if the sample contains 150 mM salt, the salt carries it: the voltage never climbs to where focusing happens, the strip heats, and the ends of the gradient burn.
4The two blind spots, and the bottleneck
- Basic proteins
- Roughly a third of a proteome has a pI above 9. Those proteins focus at the extreme basic end, where electro-osmotic flow and precipitation at the pI work against them, and they are off a pH 4–7 strip altogether. Dedicated basic-range strips and different focusing conditions recover some of them, never all.
- Membrane proteins
- A hydrophobic protein is least soluble at its own isoelectric point, which is exactly where focusing takes it. Many precipitate on the strip and never reach the second dimension, whatever detergent is in the rehydration buffer. This under-representation is structural, not a technique problem to be solved.
- Spot matching
- Comparing two gels means finding each spot again on the other one, and no two large-format gels have quite the same geometry. Software warps the images and matches spots; crowded regions are mis-matched, and a mis-match produces a dramatic apparent fold change because it is comparing two different proteins. This, not the staining, is what limits quantification.
- Spot volume
- Integrated stain intensity over a spot, which is the quantity people report. Its gel-to-gel coefficient of variation runs from about 20% with a fluorescent stain to 45% with silver: larger than many of the biological effects a 2D study is looking for.
5DIGE: putting the samples on one gel
Difference gel electrophoresis attacks the matching problem directly. The samples are covalently labelled before focusing with size- and charge-matched cyanine dyes (typically a few per cent of lysine residues, so the labelled and unlabelled forms co-migrate) and run together on a single gel.
6What a spot is not
A position on a 2D map is not an identification. Two proteins can share a pI and a mass, a spot in a crowded region often contains several, and modification shifts a protein away from where its sequence predicts it should be.
Key terms
Practise these as flashcards →The pH at which a protein's net charge is zero. In a pH gradient it is the position where the protein stops moving.
Immobilised pH gradient: acrylamide with buffering groups copolymerised into the matrix, so the gradient cannot drift during a long focus.
The integral of voltage over time, which is how the end of focusing is specified. Typically 40–60 kVh for an 18 cm strip.
A mixture of small amphoteric molecules added to the rehydration buffer. They improve protein solubility and smooth the conductivity through the run.
One specific molecular form of a protein from a given gene, including its modifications and processing. The horizontal train on a 2D gel is a proteoform series.
A vertical tail above a spot, caused by free cysteine thiols re-oxidising during the second dimension. Alkylation after reduction prevents it.
A pooled sample labelled with a third dye and run on every gel, so every spot on every gel is measured as a ratio to the same reference.
When to use it
- Showing that a protein's modification state changes, when you need to see the states separately rather than as a total.
- Screening for differences between two states of a sample when you want intact proteins rather than peptides, so that a hit can be picked and identified.
- Checking the charge heterogeneity of a purified protein or a biologic, where a train of spots reports deamidation or incomplete processing.
- Resolving two proteins of the same apparent mass that a one-dimensional gel reports as a single band.
Compared with alternatives
The second dimension on its own. Faster and far easier, but it adds every proteoform of a protein into one band.
Deeper, quantitative and not biased against basic or membrane proteins; prefer it for discovery, and use a 2D gel when you specifically want intact proteoforms separated.
Measures a specific modification directly with antibodies or enrichment; a 2D gel sees the charge consequence of any modification without knowing which it is.
Can be run on a 2D gel to tell you which train of spots is your protein, and is the usual way to confirm that a 2D spot is what you think.
Ion exchange separates charge variants preparatively and at scale, where a 2D gel is analytical only.
Sub-techniques
Choosing one here switches the whole page, including the protocol and the lab, to it.
The first dimension used on its own: proteins migrate in a pH gradient until their net charge is zero, which makes it a charge separation with a self-sharpening mechanism and resolution of a few hundredths of a pH unit. Run in IPG strips, in capillaries (cIEF) or as free-flow systems, it is also a fractionation step ahead of mass spectrometry.
- Resolution depends on the pH range per centimetre, so narrow-range strips resolve proteoforms that broad ones merge.
- Everything sharing a pI arrives at the same place, so on its own it resolves far fewer species than the two dimensions together.
- Capillary IEF with whole-protein detection is the standard charge-heterogeneity assay for therapeutic antibodies.