Experiment9 Separation and identification of lactate dehydrogenase ( LDH ) isoenzyme.
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Transcript of Experiment9 Separation and identification of lactate dehydrogenase ( LDH ) isoenzyme.
Experiment9 Separation and identification of lactate dehydrogenase
( LDH) isoenzyme
Master the method of agarose gel electro
phoresis to separate and identify lactate deh
ydrogenase isoenzyme.
1. Aim and request
Lactate dehydrogenase ( LDH ) catalyses
the reaction between lactate and pyruvic acid. In
alkaline buffer solution, enzyme proteins are ne
gatively charged. They will migrate toward anod
e in electric field. Because of the difference of n
et charge number, their migration velocities are
distinct.
2. Principle
The LDH with more ‘H-subunit’ will have
greater migration velocity. The LDH with
more ‘M-subunit’ will have smaller migration
velocity.
Use agarose gel plate as the supporting mediu
m and separate LDH isoenzyme by electrophor
esis in electric field. After electrophoresis, cov
er the agarose gel plate with the dye solution.
LDH catalyze the removal of hydrogen atoms f
rom lactate.
The product NADH + H+ reacts with the artificial hydrogen transmitter phenazine methosulfate(PMS) and finally with the last hydrogen acceptor nitroblue tetrazolium(NBT). NBT is deoxidized to purple compound. Then every zones of LDH isoenzyme can be seen. Estimate their relative content by darkness of color.
lactic acid
pyruvate
NAD+
NADH+H+
PMS-2H
PMS
NBT
NBT-2H(hyacinthine)
⑴ Agarose gel plate preparation. Spread 5 ml hot
agarose solution evenly onto a clean glass plate (air
bubbles must be removed), and cool it naturally. ⑵Make an opening at the site 1.5cm from the edg
e of the plate. Put the plate on the electrophoresis de
vice, add 3~5μl serum sample on the opening, turn o
n the power and let the electrophoresis continue for
30 minutes.
3. Procedure
⑶ After electrophoresis, cover the plate
with the dye solution. ( this step is finished
by the teacher).
⑷ Incubate the plate in warm bath for
several minutes until the color appears
Record what you have observed on your
lab reports.
4. Results
LDH isoenzyme in healthy adult serum has the order as follows:
LDH2 > LDH1 > LDH3 > LDH4 > LDH5
5. Reference
⑴ Describe the principle of the LDH assay.
⑵ How many bands can be visualized in
this experiment?
Questions