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From the Neurobiology Department, University Medical School, Framlington Place, Newcastle upon Tyne, United Kingdom
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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2 chain, all of the
sarcoglycans, and caveolin 3) are separated in the lower phase.
This basic system could be used for different combinations of
antibodies as new muscular dystrophy proteins are identified and
require examination. In addition, analysis of the laminin 2
chain of merosin showed that this protein was expressed as a doublet or
triplet set of bands in many patients with active muscle pathology.
This may indicate the existence of an embryonic isoform, which
is re-expressed in regenerating fibers.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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.
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2 laminin are more subtle
and difficult to detect. It would therefore be helpful if protein
analysis could be used to indicate where to start the search for gene
mutations.2
Analysis of muscular dystrophy proteins
obviously requires a muscle biopsy to be taken, but these are commonly
performed as part of the routine diagnostic procedures undertaken for
new patients presenting with muscle problems. At Newcastle we now undertake immunocytochemical and immunoblotting analysis of biopsies from all new muscular dystrophy patients using a panel of monoclonal antibodies. Our experiences with BMD demonstrated just how broad the clinical phenotype could be, and as the other recessive dystrophies are still being defined at the clinical level, we feel that it is appropriate to test all biopsies with all of the antibodies that are currently available. This sort of approach allows for unexpected diagnoses in patients with atypical clinical presentation3,4 and can sometimes lead to the definition of clinical subgroups with characteristic abnormalities of protein expression, but where the primary gene defect is still unknown.5,6
Labeling sections with a panel of antibodies is straightforward (so
long as the antibodies recognize native protein in sections), but it
would not be cost-effective to run a blot for every antibody. We have
therefore evolved a biphasic gel system, which enables the
corresponding blot to be probed with several monoclonal antibodies
simultaneously. The gel is optimized so that large proteins of more
than 200 kd (eg, dystrophin, dysferlin, and myosin heavy chain) can be
analyzed in the top part, while smaller proteins under 150 kd (eg,
calpain 3, the 80-kd fragment of laminin 2 chain/merosin, all of the
sarcoglycans, and caveolin 3) are separated in the lower phase. This
basic system could be used for different combinations of antibodies as
new muscular dystrophy proteins are identified and require analysis.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Muscle biopsies (usually from vastus lateralis) were taken from patients as part of the routine diagnostic procedure. Muscle samples from normal control subjects were also obtained, with consent, from legs amputated at the knee (gastrocnemius, soleus, etc) or abdominal operations (rectus abdominis). Diagnosis at the molecular level was primarily undertaken in the Department of Human Genetics (now the Human Molecular Genetics Unit in the School of Biochemistry and Genetics) of the University of Newcastle in Newcastle on Tyne.
Antibodies
The following mouse monoclonal antibodies were used on blots:
Dy8/6C5 (dystrophin carboxy terminus), Dy4/6D3 (dystrophin rod domain),
Ad1/20A6 (-sarcoglycan), ßSarc1/5B1 (ß-sarcoglycan), 35DAG/21B5
(
-sarcoglycan), Sarc3/12C1 (
-sarcoglycan), 43DAG/8D5
(-dystroglycan), Calp3d/2C4 (calpain 3 exon 1), and Calp3c/12A2
(calpain 3 exon 8).7
These antibodies are also available
commercially from Novocastra Laboratories, Newcastle on Tyne, UK. An
antibody to the laminin 2 chain of merosin was purchased from
Chemicon International, Harrow, UK (MAb 1922, which recognizes the
80-kd fragment), and the Transduction Laboratories antibody C38320 to
caveolin 3 was purchased from Affiniti Research Products, Mamhead, UK.
A monoclonal antibody to dysferlin (NCL-hamlet) is currently being
characterized in our laboratory, and this was also tested in this
multiplex system.
Polyacrylamide Gel Electrophoresis and Western Blotting
BioRad Protean II equipment was used to cast two 16-cm gels 1.5 mm thick. The resolving gel was poured in two phases: the first (bottom) phase was 7 cm of a plain 7% polyacrylamide gel (30% acrylamide plus 0.8% bisacrylamide cross-linker) in 0.375 mol/L Tris/HCl buffer, pH 8.8, containing 0.2% sodium dodecyl sulfate (SDS). As soon as the bottom part of the gel was poured, the rest of the gel was pumped on above it so that the two phases set together. The upper gradient phase was 4.5 cm of a 5.5% to 4% polyacrylamide gradient in the 0.375 mol/L Tris/HCl buffer containing 0.2% SDS. There was a gradient of glycerol throughout to help stabilize the phases: the 7% gel contained 10% glycerol, the 5.5% gel contained 7.5% glycerol, and the 4% gel contained 3% glycerol. After the resolving gel was set, the 2-cm-deep stacking gel consisting of 3% acrylamide in 0.125 mol/L Tris/HCl, pH 6.8, containing 3% glycerol and 0.2% SDS, was poured above the resolving gel and allowed to polymerize around the comb that was inserted to form the sample lanes (10 x 1 cm).8
The tissue samples were prepared while the gels were setting. Frozen muscle samples (~20 to 30 mg) were quickly weighed and homogenized twice for 15 seconds each in an Ultra Turrax homogenizer with 19 vol of treatment buffer containing 0.125 mol/L Tris/HCl buffer, pH 6.4, 10% glycerol, 4% SDS, 4 mol/L urea, 10% mercaptoethanol, and 0.001% bromophenol blue (final pH of treatment buffer was 6.8). Additional protease inhibitors are not required as all of the proteins present in the sample are denatured by the SDS and urea. Autolysis does not appear to be a problem, as judged by the satisfactory results obtained with calpain 3 analysis in this system.7 The samples were placed in boiling water for 3 minutes and centrifuged at 9500 x g for 3 minutes before 30-µl aliquots of the supernatants (equivalent to ~200 µg of non-collagen protein in control samples9 ) were applied to each lane. The gels were run at 21 mA overnight in a tank buffer containing 1.44% glycine, 0.3% Tris, and 0.2% SDS, using a thermocirculator set at 10°C.
The next day the gels were blotted with a limiting current of 1 A for 7 hours on to Schleicher and Schuell (Keene, NH) 0.45-µm nitrocellulose using a transfer buffer that contained 0.6% Tris, 2.88% glycine, 0.01% SDS, and 20% methanol.10 The BioRad (Richmond, CA) blotting tank contained plate electrodes and a supercooling coil. The equipment was attached to a thermocirculator set at -13.5°C. After blotting, the gels were fixed in 20% trichloroacetic acid, stained in 0.115% Coomassie brilliant blue R250 in 25% ethanol/10% acetic acid, and destained in 10% ethanol/10% acetic acid. The density of the myosin heavy chain band on the dried, post-blotted gel was used to indicate how much muscle protein (as opposed to fat and fibrous connective tissue) had been loaded in each sample lane.11
Multiplex Immunolabeling of Blots
Unreacted binding sites on the blots were blocked by incubation in
5% low-fat dried milk powder (supermarket) in a pH 8 buffer containing
10 mmol/L Tris/HCl, 0.15 mol/L NaCl, and 0.05% Tween 20 (TBST) on a
rocking table for 1 hour at room temperature. This milk solution was
retained. The primary mouse monoclonal antibodies were added together
in a cocktail. The antibodies may be used in any combination, so long
as the size of all of the potentially immunoreactive bands is taken
into consideration. Some proteins, such as dystrophin or calpain 3,
produce more than one band, and the laminin 2 chain of merosin is
detected as 80-kd or 300-kd fragments according to the antibody used.
It is also obviously not possible to analyze proteins of the same size
on the same blot. The dilutions of antibodies used are shown in Table 2
, but these suggestions are for guidance
only; higher dilutions may well give good results, depending on the
sensitivity of the visualization system used. It is possible to retain,
freeze, and reuse the antibody cocktail, but this is not a recommended
procedure, as some antibody activity is lost at each freeze/thaw cycle.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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illustrates strips from a
blot of human skeletal muscle labeled with the multiplex antibody
combinations shown in Table 2
. This demonstrates what can be achieved,
but in practice various simpler combinations of antibodies may be more
useful. Figure 2
shows a blot labeled for
the carboxy terminus of dystrophin, the laminin 2 chain of merosin,
-sarcoglycan, and -dystroglycan. Samples from normal control
subjects, shown in the lanes labeled C, demonstrate the clear
separation of the bands representing the proteins of diagnostic
importance. Lane 1 shows a patient with partial merosin deficiency, and
lane 2 shows a sample from a CMD patient where the merosin labeling is
absent. Lane 3 shows no labeling for -sarcoglycan, and this patient
was later confirmed as having a mutation in this gene. In contrast, the
patient in lane 4 shows a partial reduction in -sarcoglycan labeling
and was found to have a mutation in the -sarcoglycan gene. The
patient in lane 5 has Duchenne muscular dystrophy, and although
labeling for dystrophin is absent, labeling for all of the other
proteins is reduced too. The myosin heavy chain labeling in the bottom
panel confirms that this reduction is not simply due to insufficient
sample loading.
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is from a patient with polymyositis
and demonstrates a doublet of bands for the laminin 2 chain of
merosin. As we have used the multiplex approach to blotting, we have
seen many examples of multiple laminin 2 chain bands in patients
with muscle diseases where there is a lot of fiber degeneration and
regeneration going on. Figure 3
illustrates some of the variation observed, from overexpression in one
LGMD patient (band 7) to very weak bands in a DMD patient (band 13).
The most intensely labeled components of the doublet and triplet bands
are often lower than the band in the normal control samples (band 3,
for example). These bands are not seen in normal control muscle but do
occur in fetal tissue (results not shown). Thus, this may be evidence
for an embryonic isoform of laminin 2 chain, with re-expression in
regenerating fibers.
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illustrates the
results obtained with a group of six patients who presented with muscle
weakness in a limb-girdle distribution and high creatine kinase levels.
For this analysis, one multiplex contained antibodies to the carboxy
terminus of dystrophin, exon 1 of calpain 3, -sarcoglycan, and
ß-dystroglycan (Figure 4A)
, whereas the second multiplex contained
antibodies to the rod domain of dystrophin, exon 8 of calpain 3, the
2 chain of laminin (merosin), ß-dystroglycan, and -sarcoglycan
(Figure 4B)
. Labeling for ß-dystroglycan is useful as a control for
the diagnostic proteins and was therefore included in both. The
patients in lanes 1 and 6 remained unresolved, with band patterns that
were indistinguishable from those in normal control subjects. Normal
labeling was also observed on sections labeled with antibodies to all
of the proteins currently known to show primary or secondary
abnormalities in muscular dystrophy patients. The patient in lane 2 had
no detectable laminin 2 chain on blots (see panel A at 80 kd) and
had a pattern of muscle involvement that matched the small group of
patients previously described with this secondary abnormality of
protein expression.6
The patient in lane 3 had dystrophin
bands of abnormal size (see panels A and B at 400 kd), which are
characteristic of patients with Becker MD. The patient in lane 4 was
unresolved by this combination of antibodies, but the addition of the
dysferlin antibody to the multiplex in panel A revealed that this
patient had LGMD2B (manuscript in preparation). Finally, the patient in
lane 5 was found to have abnormal expression of calpain 3 (see panel A
at 94 kd and 30 kd and panel B at 94 kd and 60 kd), and the diagnosis
of LGMD2A was later confirmed by the finding of a mutation in the
CAPN3 gene.
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illustrates an additional band that we
have observed in several cases, all but one being in a
sarcoglycanopathy. An odd band at ~30 kd was detected in the
multiplex system, but subsequent analysis with the individual
antibodies alone showed that this band was labeled by antibody
43DAG/8D5 to ß-dystroglycan. Careful examination of our records
showed that, in every case, the sample had been sent from outside
Newcastle and that there had been some delay in transit. It appears
that this band is therefore a degradation product of ß-dystroglycan,
and it seems likely that instability of the dystrophin-glycoprotein
complex due to sarcoglycan deficiency may predispose
ß-dystroglycan to break down in this manner. A mutation in the
-sarcoglycan gene has been confirmed in the patient shown in Figure 5
.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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2 chain of merosin on
blots.6 We found that our modified methods of electrophoresis and blotting were better for analyzing the proteins in skeletal muscle samples than the classical methods of Laemmli12 and Towbin.13 Running the resolving gel slowly overnight and increasing the SDS in the polyacrylamide gel matrix to 0.2% improved the resolution of the high molecular mass proteins. Incorporating glycerol in to the stacking gel also seemed to improve the resolution of the high molecular mass bands and reduced the flare from the myosin heavy chain band (which is effectively grossly overloaded in proportion to the other muscle proteins present). The addition of 4 mol/L urea to a treatment buffer already containing 4% SDS aided the dissociation and solubilization of large protein complexes in skeletal muscle, particularly those of myosin and laminin. The addition of 0.01% SDS to the blotting buffer encouraged large proteins out from the gel matrix (but did not appear to inhibit binding to the nitrocellulose, as judged by the detection of dystrophin in DMD patients11 ), whereas the 20% methanol helped to fix the proteins to the nitrocellulose sheets.
During the development of this method we have identified two common sources of disappointing results. First, if there is a change in sample composition between adjacent lanes, one lane may appear skinny and distorted. Thus, if a sample has a low myosin content so that all of the protein bands are faint, it is not feasible to simply double the volume of sample applied. This results in an imbalance in the SDS and urea content between adjacent lanes. To analyze a sample like this, the best approach is to homogenize an additional sample with a 1:10 rather than 1:20 volume of treatment buffer. Alternatively, the sample volume may be doubled so long as an extra volume of treatment buffer is added in all of the other sample wells. Too large a volume will result in poor band resolution, however. The other common problem arises from using muscle samples that have been set in mountant for sectioning. Contamination with mountant results in band smearing, and this should be avoided by slicing as much mountant as possible off while the tissue is frozen. As a last resort the muscle sample may have to be thawed and the mountant quickly wiped off on a paper tissue before homogenization.
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Footnotes |
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Supported by grants from the Muscular Dystrophy Group of Great Britain and the Association Française contre les Myopathies.
Accepted for publication December 14, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-, ß-, and -sarcoglycan in patients with limb-girdle muscular dystrophy. Neuromuscul Disord 1996, 6:467-474[Medline]
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