Exercise 4.7 - Separation of LDH Isozymes from Serum
Figure 4.10 Preparation rack for tube gels
Figure 4.11 Filling gel tubes
Figure 4.12 Assembling tubes and bath chambers
Figure 4.13 LDH isozyme separation
Figure 4.14 Densitometry tracings of LDH patterns
- 1.0 M Tris-HCl pH 8.8
- Stock 30%T:0.8%C Acrylamide
- 10% (w/v) Ammonium persulfate
- 40% (w/v) Sucrose
- Tris-Glycine Buffer
- LDH Stain: Must be prepared just prior to use. For use,
combine 8 parts A to 1 part B.
|60% Sodium Lactate
|Nitro blue tetrazolium
|Sodium phosphate buffer to make
|0.5% (w/v) NAD (use 5 mg/ml HO)
- Non-heparanized hematocrit tubes and centrifuge
- Blood lancets and alcohol for swabbing
The following directions are given for the preparation
of tube gels. Many laboratories continue to use tube gels.
The directions can be readily modified to use slab gels as
detailed in Exercise 4.1
and Exercise 4.2
- Assemble the appropriate sized glass tubes and a
preparation rack. These may be cut from standard glass
tubing or available commercially.
- Insert the glass tubes in the preparation rack by laying
a strip of Parafilm approximately 14" long on top of the
black grommets located on the base of the preparation rack
(Figure 4.10). Place Parafilm so that it just covers the
first grommet and allow an approximate 2" overhang at the
opposite end. Beginning at the first grommet, insert the
tube through the hole in the upper section of the rack and
press the tube down into the grommet, pushing the Parafilm
ahead to form a tight leak-proof seal. Place your finger
under the grommet and seat the tube and Parafilm against
your finger. Do not stretch or pull the Parafilm while
inserting the tubes. Leave a small amount of slack as you
proceed from one tube to the next.
- Prepare the separating gel (5% acrylamide) by mixing the
Mix slighty more than 1 ml of solution for each tube being
prepared. Once the reagents are mixed, complete the next two
steps quickly. The mixture will begin to polymerize in
approximately 10 minutes. Rinse syringe, beakers, and other
implements after use to prevent the gel solution from
solidifying on them.
- Using a 10 cc syringe with a Loading Syringe Stub
Needle, fill the gel tubes to within approximately 2 cm of
the top with the separating gel mixture (Figure 4.11).
Expel any remaining solution from the syringe when done.
- Insert the syringe with stub needle into each gel tube
as far as it will go; draw off as much gel solution as
possible. This procedure will result in gels of uniform
height. After removing excess gel solution from the gel
tubes, rinse the syringe and stub needle.
Carefully add a small layer of Tris-HCl Buffer to the top of
the gel, without disturbing the gel itself.
- Allow the separating gel solution to stand undisturbed
for 30 minutes during polymerization.
The following step involves the use of human blood. Extreme
caution must be taken to guard against the dangers of
infection. Use disposable lancets, wear gloves at all times
and dispose of sharps as indicated by the instructor.
Alternatively use blood from a laboratory animal.
- Using a non-heparanized hematocrit capillary tube,
obtain a sample of blood. Centrifuge the blood sample in a
hematocrit head of a clinical centrifuge to separate the
formed elements from the serum/plasma. A standard hematocrit
tube contains just the right serum for a single gel tube
analysis. Alternatively, blood can be centrifuged in regular
centrifuge tubes, if there is a sufficient quantity of
- For each blood sample, prepare a 1:10 dilution of the
sample by combining 1 part of sample serum with 9 parts of
sucrose. Prepare at least 100 µ l of diluted
serum for each specimen, enough for two gels.
- Remove the storage solution from three gel tubes by
inverting the gel tubes over a layer of absorbent paper.
Shake the tubes abruptly once or twice in a downward motion
to remove all the buffer. While the gel tubes are still
inverted, blot the ends of the tubes to remove any remaining
- Apply 50 µ l of diluted serum to each of
two gel tubes. Do not touch the surface of the gel, but
allow the sample to flow onto its surface.
- Layer each gel tube with tris-glycine buffer to
completely fill the tube. Be extremely careful not to
disturb the serum layer.
- Remove gel tube #1 from the preparation rack. Moisten
the upper end of the tube with a little water and insert
sample end up into the bottom of the bath tube adapter
(position 1 -- ). Push the tube in so that its
upper edge is flush with the upper edge of the adapter.
Place all gel tubes into the bath in this manner. Observe
the numbers on the upper bath lid and use them to identify
the corresponding samples from the preparation rack. Handle
the gel tubes with care to prevent mixing of sample and
buffer layer. Use the hollow plastic bath stoppers to plug
any empty tube adapters.
- Pour enough tris-glycine buffer into the lower bath so
that the bath is approximately half full.
- Assemble the upper and lower chambers, ensuring that the
lower portions of the gel tubes are immersed in the lower
bath and that all trapped air bubbles are removed from the
ends of the tubes.
- Carefully pour additional tris-glycine buffer into the
upper bath, so that the level is sufficient enough to make
contact with the cathode when it is inserted (Figure 4.12).
Be particularly careful not to disturb the buffer inside the
gel tubes. Again, check the gel tubes to insure that there
are no air bubbles trapped in the upper part of the gel
- Place the cover onto the upper bath and connect the
electrodes to the baths and the power supply.
- Turn on the power source and, if necessary, allow it to
warm up. Check the bath to insure that the polarity is
normal. Connect the safety interlock jack to the pins on
Adjust the power source to deliver 5 milliamps of current
per gel tube in the bath. For example, if 6 gel tubes are
being run, the total current should be 30 milliamps.
- Continue electrophoresis for 20 minutes or until a
clearly defined albumin band is seen near the bottom of the
tube. If prestained protein markers are used in the
standard, the timing can be precisely controlled by visually
checking its progress.
- When electrophoresis is complete, disconnect the safety
inter lock and turn the power source off. Pour the upper
bath buffer into a storage container. Place the upper bath
on the U-stand and remove the first gel tube.
- Remove the separating gel for staining. Fit the 10 cc
syringe with the blunt-tipped gel removing needle and half
fill the syringe with water. While holding the gel tube with
one hand, carefully insert the needle at the sample
application end of the tube, between the inside wall of the
tube and the gel. While slowly pushing the needle in and
keeping it flat against the tube wall to avoid scratching
the gel, force a stream of water through the needle and
rotate it completely around the circumference of the gel.
Remove the needle and insert it from the other end of the
tube (the separating gel end) using the same technique
described above. Once the needle is inserted and rotated
completely, the gel will come loose and slide from the tube.
- Rinse the gel with distilled or deionized water to
remove any enzymes on the surface and place the gel in a
stoppered test tube.
- Repeat steps 21-22 for the rest of the tubes. Label each
- Place 2 ml of freshly prepared LDH stain solution into
10 x 75 mm amber tubes and add a gel to each tube. Keep
stoppered and away from light.
- Incubate the gels in stain solution at 37 ° C for 60 minutes.
- When the color of the bands has developed, drain off the
stain solution from the tubes and fill them with 7% acetic
acid. Continue to protect the gels from light for one hour.
At the end of this time, the acetic acid will have inhibited
further color development and preserved the protein gels.
- Transfer each gel to a clear glass test tube containing
fresh 7% acetic acid and stopper the tube. The gels are now
ready for photography or densitomer quantitation. Figure 4.13
demonstrate LDH separation patterns for a normal individual
and a comparison with a pattern consistent with myocardial
- If the gels are scanned, integrate the area of each band
and calculate the amount of each isozyme as a % of the total
The term isozyme was introduced by C.L. Markert and F.
Moller in 1959
to describe multiple enzyme forms with similar or identical
substrate specificity, and occurring within the same
organism. Markert later proposed to modify the term by such
adjectives as allelic, nonallelic, multimeric,
conformational, and conjugated. These adjectives imply a
broader definition of the term isozyme and thus include many
genetic variations, as well as physiological modifications
of the protein structures.
Serum lactic dehydrogenase (LDH) is an ideal enzyme for
the analysis of isozymes, and is also a model system for
electrophoretic analysis. The enzyme actually consists of
five electrophoretically separable isoenzymes, identified as
LDH--1, LDH--2, LDH--3, LDH--4, and LDH--5 in the order of
their relative rates of electrophoretic migration; LDH--1
migrates most rapidly. Since these isoenzymes are usually
associated with characteristic tissue or organ sources,
their relative concentrations in serum may provide useful
information in the differentiation of diseases of various
body systems, such as myocardial infarction, liver necrosis,
pulmonary infarct, primary muscle dystrophy, pernicious
anemia, and malignancy. Consequently several easily
available commercial kits can be used for their analysis.
LDH isozymes are important indicators of cellular
differentation as well. Analysis of the peptide synthesis
for LDH isozymes presents what is now a classical analysis
of differential gene activity.
LDH1 is composed of a single peptide species A, while
LDH5 is composed of the single peptide species B. LDH2-4 are
the permutations of combining species A and B into a
functional tetramer. Thus, whether LDH1 or LDH5 are
synthesized depends entirely on the gene transcription and
translation for species A and B respectively. If both are
turned on and are equimolar within the cell, then LDH3
will be the predominant form (AABB). In short, the LDH
tetramer is a self-assembling molecule that depends upon the
concentration of reactants for its tetrameric form. The
distribution of isozymes can thus be analyzed statistically
to determine the extent to which gene A and/or gene B are
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Cell Biology Laboratory Manual
Dr. William H. Heidcamp, Biology Department, Gustavus Adolphus College,
St. Peter, MN 56082 -- firstname.lastname@example.org