SDS-PAGE is an electrophoresis method that separates proteins by molecular weight and presents the result as visible bands. Its name comes from the initials of the detergent used and the separation medium: sodium dodecyl sulfate and polyacrylamide gel electrophoresis. Because it is the most practical way of seeing how many weight groups a protein mixture falls into, it is widely used in laboratories.
The method has held its place despite being in use for decades. It is relatively simple to set up, needs no expensive instrument and gives a result that can be assessed directly by eye. A first impression of a sample's purity, or of whether it contains an unexpected component, most often comes from this method.
Principle of the method
In their native state, proteins are folded in different shapes and carry different net charges. These two variables make it impossible to separate them by size alone. SDS solves the problem in two ways: it unfolds the molecule, doing away with the folding, and it binds along the chain in proportion to mass, giving every protein a similar negative charge density. Movement in an electric field then depends essentially on the size of the molecule.
The separation medium is a porous mesh of polyacrylamide. Small molecules pass easily through this mesh and travel far, while large ones are held back by friction. The result is an approximately linear relationship between migration distance and the logarithm of molecular weight. The acrylamide percentage of the gel sets the pore size: high-percentage gels resolve small proteins better, low-percentage gels large ones.
In practice the gel is usually prepared in two layers. The loose upper layer gathers the proteins coming from the different wells into a thin line; the separation itself takes place in the tighter lower layer. This arrangement gives sharp bands. Current and run time are set together so that the gel does not heat up during the run.
The steps of a run
- The sample is mixed with detergent and loading buffer; if a reducing component is added, the disulfide bridges are opened.
- The mixture is heated briefly to complete denaturation.
- The samples and a standard ladder are loaded into the wells of the gel.
- An electric field is applied; because the proteins are negatively charged, they migrate towards the positive pole.
- Staining is carried out; the common option is a protein stain that colours the gel blue, while more sensitive methods are used for low amounts.
- Excess stain is removed and the gel is imaged so that the band patterns can be assessed.
The choice of stain determines the lowest amount that can be seen. Common blue stains are practical and cheap but may miss bands at low concentration; more sensitive methods make small amounts of a component visible, though they are laborious to apply. For this reason, the absence of a band does not mean that no component of that weight is present.
The ladder and reading weights
A standard ladder is a mixture of proteins of known molecular weight and forms a reference ruler on the gel. The approximate weight is determined by comparing the position of the sample bands with this ruler. In the reference batch analysis, a ladder covering the range from 10 to 250 kilodaltons was used, the sample's main protein density was observed in the 10-25 kilodalton range and a distinct band was recorded at around 37 kilodaltons.
| Observation | Possible meaning |
|---|---|
| A sharp, single band | A dominant component or a narrow weight group |
| Many faint bands | The mixture is spread over a wide weight range |
| A diffuse band | Overloading or a problem with the run conditions |
| Material collecting at the bottom of the well | Undissolved or aggregated material |
| Absence of a band | Insufficient loading, or falling below the sensitivity of the stain |
The amount loaded also affects the result. In the reference batch analysis the sample was loaded as a series ranging from 5 to 30 micrograms, so that the range in which faint bands become visible and dense bands do not overflow could be observed.
Points to watch with venom samples
The peptides of interest in venom are very small and stay close to the lower limit on standard gels; gels with a high acrylamide percentage, or buffer systems suited to small molecules, are therefore preferred. The second point is the reduction step: because opening the disulfide bridges can change a peptide's migration behaviour, conditions with and without a reducing agent are assessed separately. The role of the bridges in the structure is explained in the article on disulfide bridges. So that the loading amount can be chosen correctly, the total protein content of the sample has to be measured beforehand; that measurement is covered in the article on protein assay with a standard curve.
When the result is reported, the run conditions are given alongside the gel image: the acrylamide percentage of the gel, the ladder used, whether reduction was applied and the amount loaded. Without this information, a band pattern cannot be compared with the result of another study. Keeping the image scaled and unprocessed is also necessary for assessments made later.
What the method does not tell you
What electrophoresis gives is an apparent molecular weight, not an exact mass. Charge distribution, residual folding or sugar groups attached to the molecule can affect migration and produce a position different from the one expected. More than one component may also be present within the same band. For exact mass and sequence information, methods such as mass spectrometry are used. Electrophoresis is therefore used not as a test that decides on its own, but as one layer read together with chromatography and spectroscopy. The points where the three methods confirm one another form the basis of the assessment given for a batch; the points where they diverge are grounds for repeating the measurement. Batch-level results are published on the analysis page.
