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Isoelectric focusing is used to resolve the
isoenzymes of GALT. The band patterns, when used in
conjunction with a quantitative GALT result, can be used to predict
the GALT phenotype of an individual.
In isoelectric focusing, a pH gradient is established across an
agarose gel by adding a select mixture of amphoteric molecules
to the gel and applying an electric field to the gel. Each protein
(isoenzyme) has its own unique isoelectric point, a pH at which the
net charge of the protein is equal to zero. Therefore, if a protein is
applied to the gel, it will migrate through the pH gradient in the gel
until it reaches its isoelectric point. There the protein will stop and
"focus" into distinct bands.
In this procedure, a red blood cell hemolysate is focused on a
5% agarose gel containing ampholytes of a 5-7 pH range.
The isoenzyme bands are then visualized by applying a substrate
mixture that results in a series of reactions (shown below).
The final product, NADPH, is stained a blue-violet color when it
reacts with phenazine methosulfate (PMS) and 3-(4-5
dimethylthiazol-2-yl)I-2,5-diphenyltetrazolium bromide (MTT).
(Shin YS, Niedermeier HP, Endres W, et al: Agarose gel
isoelectrofocusing of UDP-galactose pyrophosphorylase and
galactose-1-phosphate uridyltransferase: developmental aspect
of UDP-galactose pyrophosphorylase. Clin Chim Acta 1987;166:
27-35, modified to acrylamide as described by Leclerc P, Forest
JC: Electrophoretic determination of isoamylases in serum with
commercially available reagents. Clin Chem 1982;28:37-40)
GALT
Gal-1-P UDP-Glu -------------------> UDP-Gal Glu-1-P
Phosphoglucomutase
Glu-1-P Glu-1-6 diP --------------------> Glu-1-6 diP Glu-6-P
Glu-6-P-Dehydrogenase
Glu-6-P NADP -----------------------> Ribose-5-P CO(2) NADPH
NADPH MTT PMS ---------------> Blue Violet Stain