Agriculture Reference
In-Depth Information
Polyacrylamide gels are composed of acrylamide plus a cross linker, usually bis-acrylamide, together
producing mesh-like polyacrylamide gels. Different concentrations of acrylamide produce different pore
size gels capable of separating proteins according to molecular weight differences. The most commonly
used technique is sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). SDS is a dena-
turing detergent that binds to proteins, and in so doing unfolds the polypeptides and confers a negative
charge on them in proportion to their length. Because all polypeptides become negatively charged, separa-
tions in an electrical ield will be solely due to size differences of polypeptides. Following electrophoresis,
the gel is stained, usually with either Coomassie Brilliant Blue or silver stain. Staining allows for the visu-
alization of the separated proteins, and the variation in protein banding patterns can be related to varietal
differences. As with isozyme analysis, Rf values determined using reference proteins, can be calculated
after the protein bands are stained to aid in the identiication of speciic bands.
A less commonly used method for varietal testing is native gel electrophoresis. In this method, no
denaturing agent such as SDS is used. Proteins therefore remain in their “native” state, and therefore separa-
tions are based on charge-to-mass ratio differences rather than size alone. Since separated proteins remain
in their native state, they can be visualized using either general protein staining reagents or by more speciic
enzyme-linked staining. Another more recent application has been capillary zone electrophoresis which
is similar in principle to high performance liquid chromatography. This approach provides rapid analyses
(within 10 minutes) with high resolution for a wide range of compounds although the initial cost of equip-
ment is high. Electrophoretic approaches to varietal identiication testing are considered by Lookhart and
Wrigley (1995).
Two dimensional Electrophoresis-Isoelectric focusing
Although SDS-PAGE is the most common protein electrophoresis technique for varietal testing, other
electrophoretic methods have been used. Two-dimensional (2-D) electrophoresis is a technique capable
of separating extremely complex protein mixtures. Two-dimensional electrophoresis consists of a tandem
sequence of electrophoretic separations. In the irst dimension, called isoelectric focusing, proteins are sep-
arated in a polyacrylamide gel with a pH gradient and an electrical ield applied across the gel. Since pro-
teins are charged molecules, they will migrate towards either the more negative or positive end. Migration
will stop when a protein reaches its isoelectric point, the pH at which that protein has no net charge. After
this separation based on isoelectric point, a second separation (second dimension) takes place at a 90-degree
angle from the irst. In this second dimension, proteins are separated according to molecular weight using
SDS-PAGE, as described above. By using two separation properties rather than just one, proteins can be
more effectively separated by 2-D electrophoresis.
Electrophoresis Check Samples
It is essential that appropriate check samples be tested along with a test sample while using any of the elec-
trophoretic procedures. The banding patterns of the samples being tested should be compared with those of
the check sample. The use of a check sample for comparative purposes will ensure that any observed dif-
ferences have a genetic basis and are not due to the testing procedure. Because electrophoresis procedures
are varied and continue to undergo reinement, detailed descriptions of electrophoresis procedures will not
be presented here. Interested readers are encouraged to review the AOSA Cultivar Purity Testing Handbook
and the ISTA recommended procedures for polyacrylamide gel electrophoresis and the Seed Technologist
Training Manual.
