Development and Validation of a Western Blot Method to Quantify Mini-Dystrophin in Human Skeletal Muscle Biopsies.
Development and Validation of a Western Blot Method to Quantify Mini-Dystrophin in Human Skeletal Muscle Biopsies.
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DOI：
10.1208/s12248-022-00776-0
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发表时间：
2022-12-20
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杜氏肌营养不良症 (DMD) 是一种退行性肌肉疾病，大约每 5000 名男性出生时就有一人患有这种疾病。该疾病通常是由抗肌营养不良蛋白基因中遗传性 X 连锁隐性致病变异引起，但也可能由新生突变引起。引起疾病的变异包括无义、框外删除或重复，导致肌营养不良蛋白表达丧失。目前 DMD 尚无治愈方法，并且少数可用的治疗方案旨在减缓肌肉退化。基因治疗的新进展以及对其他肌营养不良症中肌营养不良蛋白 (DYS) 表达的了解为治疗开辟了新的机会。因此，需要可靠的方法来监测肌营养不良蛋白的表达并评估新疗法对DMD和贝克尔肌营养不良症（BMD）等肌营养不良症的疗效。在这里，我们描述了一种新的蛋白质印迹（WB）方法的验证，该方法用于定量人类骨骼肌组织中的微型肌营养不良蛋白，该方法易于在大多数实验室环境中采用。该WB方法通过精密度、准确度、选择性、稀释线性、稳定性和重复性进行评估。基于 mini-DYS 标准性能，该检测的动态范围为 0.5–15 ng 蛋白（每泳道每 5 μg 总蛋白），精密度为 3.3% 至 25.5%，准确度为 − 7.5% 至 3.3%。我们的稳定性评估表明，该蛋白质在 4 个 F/T 循环后、室温下长达 2 小时以及 − 70°C 下 7 个月后保持稳定。此外，我们的 WB 方法与我们最近发布的 LC-MS 方法的结果进行了比较。
Duchenne muscular dystrophy (DMD) is a degenerative muscular disease affecting roughly one in 5000 males at birth. The disease is often caused by inherited X-linked recessive pathogenic variants in the dystrophin gene, but may also arise from de novo mutations. Disease-causing variants include nonsense, out of frame deletions or duplications that result in loss of dystrophin protein expression. There is currently no cure for DMD and the few treatment options available aim at slowing muscle degradation. New advances in gene therapy and understanding of dystrophin (DYS) expression in other muscular dystrophies have opened new opportunities for treatment. Therefore, reliable methods are needed to monitor dystrophin expression and assess the efficacy of new therapies for muscular dystrophies such as DMD and Becker muscular dystrophy (BMD). Here, we describe the validation of a novel Western blot (WB) method for the quantitation of mini-dystrophin protein in human skeletal muscle tissues that is easy to adopt in most laboratory settings. This WB method was assessed through precision, accuracy, selectivity, dilution linearity, stability, and repeatability. Based on mini-DYS standard performance, the assay has a dynamic range of 0.5–15 ng protein (per 5 μg total protein per lane), precision of 3.3 to 25.5%, and accuracy of − 7.5 to 3.3%. Our stability assessment showed that the protein is stable after 4 F/T cycles, up to 2 h at RT and after 7 months at − 70°C. Furthermore, our WB method was compared to the results from our recently published LC–MS method.
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具有未验证抗体的蛋白质印迹不准确：需要蛋白质印迹最小的报告标准（WBMR）。
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