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(PAGE)
Electrophoresis in a polyacrylamide matrix
separating or resolving molecules in a mixture
under the influence of an applied electric field
PAGE used for proteins and small pieces of DNA
Similar idea to separation of DNA in agarose
2 major determinants of particle mobility are
charge/mass ratio and structure
Polyacrylamide has smaller pore size- better
resolution (2bp differences in DNA) and
separates smaller molecules (50-500bp)
One bp has molecular mass of 660 Daltons
(Dalton=gm/mole)
1 kb piece of DNA = 660kDa
Average protein has mass of ~ 60kDa.
PAGE used for proteins and small pieces of DNA
Acylamide is synthesized (not natural like agarose)
A typical setup consists of a gel slab sandwiched between two
glass plates, with the ends enclosed in upper and lower
reservoirs of buffer
Samples to be run are loaded in wells at the top of the gel, in
conjunction with tracking dye. An electrical voltage is applied
between the upper and lower reservoirs, causing the samples
to migrate down through the gel.
Once a gel has been 'run', it is treated to reveal the positions of the
samples
Staining
Coomassie blue-sensitive to 0.1ug of protein
Silver- sensitive to 0.002ug of protein, based on ppt of silver ions
producing brown stain, laborious.
greater sensitivity, radioactive samples can be used, allowing for
exposure over time to produce images on photographic film, as seen in
the sequencing gel on the right