SDS-PAGE versus isoelectric focusing and two-dimensional gels
SDS-PAGE (which uses a soap-like detergent) sorts proteins in a mesh-like gel by size, isoelectric focusing sorts them by charge, and two-dimensional electrophoresis uses charge first and then size.
Remember
SDS masks a protein's own charge, so SDS-PAGE sorts by size; isoelectric focusing sorts by charge; two-dimensional electrophoresis uses charge first and size second.
Protein gel electrophoresis moves charged proteins through a gel, which is a matrix that allows separation of the protein sample, in an electric field. Proteins can be separated by size, shape or charge, depending on the technique used. Three main variations exist, and the property each one uses is the key to telling them apart.
SDS-PAGE stands for sodium dodecyl sulphate polyacrylamide gel electrophoresis. SDS is a detergent (a soap-like molecule) that unfolds proteins and gives them a similar negative charge for their mass, which masks the differences in their own natural charge. The polyacrylamide gel is a mesh-like jelly, so it works as a sieve: smaller proteins travel further and larger ones lag behind. In SDS-PAGE, size is what decides how far a protein travels.
Isoelectric focusing places proteins in a pH gradient (a gradual change from acidic to alkaline). A protein's net charge depends on the pH around it, so each protein moves until it reaches the pH at which it has no net charge. That pH is its isoelectric point (pI). With no pull towards either electrode, the protein stops and forms a sharp band, so the separation depends on charge, not size.
Two-dimensional electrophoresis (2D-PAGE) separates proteins in two steps, using two different properties one after the other. Isoelectric focusing comes first, then SDS-PAGE separates the same proteins by size. A pair of proteins that overlap in the first dimension can be told apart in the second, so very complex mixtures such as serum can be resolved. That makes it useful for studying disease states in different tissues.
Key facts
- SDS-PAGE
- separates proteins mainly by size, because SDS masks their natural charge differences.
- Isoelectric focusing
- separates proteins by charge, each one stopping at its isoelectric point (pI), the pH at which it has no net charge.
- Two-dimensional electrophoresis
- uses isoelectric focusing first and SDS-PAGE second, so charge is used before size.
- Distance travelled
- in SDS-PAGE, smaller proteins move further through the gel than larger ones.
- Native PAGE
- runs proteins without SDS, so they keep their natural charge and folded shape, and their movement depends on a mixture of charge, size and shape.
- Western blotting
- detects chosen proteins with antibodies after a gel has done the separating, so it is not itself a separation.
Exam traps
- Size separation (SDS-PAGE) before isoelectric focusing reverses the order of two-dimensional electrophoresis, which uses charge first and size second.
- Native gel electrophoresis, which keeps proteins folded, does not separate mainly by size, because net charge, size and shape all influence how the proteins move.
- Western blotting is a detection method, not a separation: a gel does the separating first, and antibodies then find one chosen protein.
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