Editing Western blot (section)

=== Gel electrophoresis ===
{{Main|Gel electrophoresis}}
[[File:SDS-PAGE Electrophoresis.png|thumb|SDS-PAGE electrophoresis|upright=1.5]]
The proteins of the sample are separated using [[gel electrophoresis]].  Separation of proteins may be by [[isoelectric point]] (pI), [[molecular weight]], electric charge, or a combination of these factors.  The nature of the separation depends on the treatment of the sample and the nature of the gel.

By far the most common type of gel electrophoresis employs [[polyacrylamide]] gels and buffers loaded with sodium dodecyl sulfate (SDS). SDS-PAGE (SDS-polyacrylamide gel electrophoresis) maintains polypeptides in a denatured state once they have been treated with strong [[reducing agent]]s to remove [[Secondary protein structure|secondary]] and [[tertiary structure]] (e.g. disulfide bonds [S-S] to sulfhydryl groups [SH and SH]) and thus allows separation of proteins by their [[molecular mass]].  Sampled proteins become covered in the negatively charged SDS, effectively becoming [[anionic]], and migrate towards the positively charged (higher voltage) anode (usually having a red wire) through the [[acrylamide]] mesh of the gel. Smaller proteins migrate faster through this mesh, and the proteins are thus separated according to size (usually measured in kilodaltons, [[kDa]]). The concentration of acrylamide determines the resolution of the gel – the greater the acrylamide concentration, the better the resolution of lower molecular weight proteins.  The lower the acrylamide concentration, the better the resolution of higher molecular weight proteins. Proteins travel only in one dimension along the gel for most blots.

Samples are loaded into ''wells'' in the gel. One lane is usually reserved for a ''marker'' or ''ladder'', which is a commercially available mixture of proteins of known molecular weights, typically stained so as to form visible, coloured bands. When [[voltage]] is applied along the gel, proteins migrate through it at different speeds dependent on their size.  These different rates of advancement (different [[electrophoretic mobility|''electrophoretic mobilities'']]) separate into ''bands'' within each ''lane''. Protein bands can then be compared to the ladder bands, allowing estimation of the protein's molecular weight.

It is also possible to use a [[Two-dimensional gel electrophoresis|two-dimensional gel]] which spreads the proteins from a single sample out in two dimensions. Proteins are separated according to [[isoelectric point]] ([[pH]] at which they have a neutral net charge) in the first dimension, and according to their molecular weight in the second dimension.