Chapter 6 – Forces  245

weight, such that higher-​mass molecules appear as a band toward the top of the gel, whereas

lower-​mass molecules appear as a band toward the bottom end of the gel (Figure 6.9). Note

that enhanced separation of proteins (e.g., with a higher molecular weight cutoff) is also pos­

sible using gel electrophoresis in rationally designed microfabricated arrays, instead of the

standard slab-​cast gel systems.

Double-​stranded DNA samples are often run on agarose gels in a native state. Single-​

stranded nucleic acids, either single-​stranded DNA or RNA, have a propensity to form a range

of secondary structures in vitro due to transient Watson–​Crick base pairing interactions,

resulting in a range of mobility during gel electrophoresis. This presents potential problems

in interpretation, and so single-​stranded nucleic acid samples are often denatured first using

urea or formamide treatment.

In native gel electrophoresis, the proteins in the sample are not denatured. Such gels

can discriminate molecular components on the basis of shape, net electrical charge,

and molecular weight and are often run using 2D gel electrophoresis (2-​DE). In 2-​DE, a

population of native molecules (usually proteins) in an in vitro sample are first separated

electrophetically by their mobility on a gel that contains a pH gradient. A molecule will

therefore migrate down the electric field to the position on the gel equivalent to an overall

zero charge of that molecule at that particular pH in the gel, that is, their isoelectric point

(see Chapter 2). The sample is then treated with SDS to denature the proteins and an electric

field generated at 90° to the original field to induce electrophoretic movement horizontally

as opposed to vertically. Thus, instead of a 1D ladder of bands separating different molecules

with different molecular weights, there are 2D blobs whose position on the gel, after given

electrophoresis times in both dimensions, can be related to their molecular weight and

native electrical charge properties, with the caveat that it is a technically more challenging

and time-​consuming technique.

Molecules can be visualized on a gel using chemical staining, either directly in visible light

(e.g., Coomassie Blue is a standard stain of choice for proteins, though silver staining may also

be applied if greater sensitivity is required) or using fluorescence emission via excitation of a

stain from ultraviolet light (e.g., ethidium bromide stain, used for nucleic acids). Each band/​

blob may also be carefully extracted to reconstitute the original, now purified, sample. Thus,

FIGURE 6.9  Gel electrophoresis methods. (a) The detergent sodium dodecyl sulfate (SDS) is

used to denature and linearize proteins—​native proteins are coated in positive and negative

charge, as well as having regions of the protein stabilized by nonelectrostatic forces, such as

hydrophobic bonding (marked here with “H”). SDS eradicates hydrophobic binding and coats

all the peptide chain uniformly with them with negatively charged sulfate groups. (b) These

linearized and negatively charged proteins then migrate down an E-​field gradient, such as in

SDS polyacrylamide gel electrophoresis (left panel) here shown with molecular weight lines on

the left on the scanner gel, with proteins stained to reveal their location on the gel at a specific

time point after starting the electrophoresis in distinct bands (the different channels here show

various different mixtures of muscle proteins). The proteins in such gels may also be blotted

onto a separate substrate and probed with a specific antibody, which only binds to a specific

region of a particular protein, called a “western blot” (right panel).