Download Polyacrylamide gels

Polyacrylamide gel electrophoresis
(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.
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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
•
To calibrate the relative migrations of molecules of different size, a
marker lane is often added, where samples of known size will migrate
to reference positions
4 components of gel
1.
2.
3.
4.
Acrylamide
APS
TEMED
Bisacrylamide
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Acrylamide forms long polymer chains
Polymerization induced by APS (ammonium persulphate) which
generates free radicals (charged oxygens)
TEMED is a free radical stabilizer (N’N’N’N’-tetra methylene
diamine)
Air inhibits polymerization as it scavenges free radicals
Bis acrylamide is a cross linking agent and links long polymers of
acrylamide (N, N’-methylene bisacrylamide)
Pore size is determined by % acrylamide and the amount of cross
linker
The copolymerization of acrylamide with methylenebisacrylamide
produces a mesh-like network in three dimensions, consisting of
acrylamide chains with interconnections formed from the
methylenebisacrylamide
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SDS PAGE
• SDS is used in the gel mix.
• SDS is –ve charged and binds to proteins,
it denatures (unfolds) proteins and gives a
net negative charge.
• Proteins will then migrate to the anode
• Proteins all have same charge to mass
ratio
• Can be separated based on size alone
Native PAGE
• No denaturing agents
• Proteins separated based on size charge
and shape.
• Used when want to keep protein active to
study conformation, self-association or
aggregation, and the binding of other
proteins
Discontinuous versus Continuous
1. Discontinuous
– Common
– Better resolution
– Stacker-used to stack proteins into thin band so have same
starting position when enter separating gel. Proteins gel
squished between “leading” and “trailing” ions in the gels make
with different buffers
– Resolving gel- higher % used to separate proteins, a pH change
makes ions from the stacking gel move past the proteins and the
proteins are no longer squished.
2. Continuous
• no stacker, poor resolution, big blobs
• easier to pour
• Faster to run
Potential problems with Polyacrylamide gels
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Underloaded (bands invisible)
Sloppy loading or to little concentration of protein
Bent bands
Tearing
frowning
• Cleanliness is next to godliness when making
gels
1. Over loaded
3. Frowning (run too hot)
4. Bent bands. Tearing
2. Tearing
5. Sloppy loading or
too low conc.