Multiplex Western blotting system for the analysis of muscular dystrophy proteins

Abstract

A multiplex system of Western blotting is presented in which most of the current muscular dystrophy proteins can be analyzed simultaneously on one pair of blots. This represents a significant improvement in efficiency and cost for this type of analysis. The final diagnosis is more quickly achieved in patients where several possible diagnoses are indicated after clinical appraisal, and those with unusual presentations may be quickly resolved. The method uses a biphasic polyacrylamide gel system, which enables the corresponding blot to be probed simultaneously with a cocktail of monoclonal antibodies. 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 alpha2 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 alpha2 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.

Figure 1.

Strips from a Western blot of human skeletal muscle labeled with antibodies to the proteins indicated. The antibody combinations and dilutions are detailed in Table 2 ▶ . The left-hand strip is labeled with multiplex A, and the right-hand strip is labeled with multiplex B. Many dystrophin metabolites are labeled, as are various calpain 3 bands. The unidentified band at 120 kd is an additional singlet/doublet labeled by antibody 35DAG/21B5 to γ-sarcoglycan.

Figure 2.

Blot labeled with antibodies to the carboxy terminus of dystrophin (400 kd), the laminin α2 chain of merosin (80 kd), α-sarcoglycan (50 kd), and β-dystroglycan (43 kd). Lane C, normal control muscle samples; lanes 1 and 2, samples from patients with congenital muscular dystrophy; lanes 3 and 4, samples from patients with sarcoglycanopathies; lane 5, sample from a patient with Duchenne muscular dystrophy; lane 6, sample from a patient with polymyositis. The bottom panel shows the corresponding myosin heavy chain bands on the post-blotted gel, stained with Coomassie blue.

Figure 3.

Close-up of the 80-kd laminin α2 chain/merosin band (Chemicon MAb 1922) in samples on three blots. Bands labeled 1, 5, 6, 9, 11, and 14 are from normal control muscle samples. Samples 2, 3, and 4 are from patients with inflammatory muscle disease (polymyositis and dermatomyositis); 7, 8, 10, 12, and 14 are from patients with forms of limb-girdle muscular dystrophy; 13 is from a patient with Duchenne muscular dystrophy, and 15 is from a patient with faciosculohumeral muscular dystrophy.

Figure 4.

Immunoanalysis of muscle homogenates from six patients presenting with possible limb-girdle muscular dystrophy. A: Blot labeled with antibodies to the carboxy terminus of dystrophin (400 kd), exon 1 of calpain 3 (94 and 30 kd), α-sarcoglycan (50 kd), and β-dystroglycan (43 kd). B: Blot labeled with antibodies to the rod domain of dystrophin (400 kd), exon 8 of calpain 3 (94 and ∼60 kd), the laminin α2 chain of merosin (80 kd), β-dystroglycan (43 kd), and γ-sarcoglycan (35 kd). The 120-kd band (present as a doublet in these patients) is labeled by the antibody to γ-sarcoglycan. The corresponding myosin heavy chain bands on the post-blotted gel, stained with Coomassie blue, are shown at the bottom.

Figure 5.

Lanes from a Western blot of skeletal muscle showing muscle from two normal control subjects (C) and a patient (P) with γ-sarcoglycanopathy (limb-girdle muscular dystrophy type 2C). In the patient the labeling for dystrophin is normal, the laminin α2 chain of merosin is represented by a triplet with the middle band being most intensely labeled, α-sarcoglycan labeling is reduced, and β-dystroglycan is partially broken down to a 30-kd fragment.