The answer depends on the question: Optimal conditions for western blot characterization of muscle collagen type 1 depends on desired isoform

V. Iannarone, G. Cruz, B. Hilliard, M. Barbe
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引用次数: 11

Abstract

Fibrillar collagen type 1 is the most abundant type of collagen within the body and is a critical component of extracellular infrastructure. In order to assess collagen synthesis and extracellular accumulation in fibrotic disorders, improved methods are needed to detect changes in procollagen versus mature collagen at the protein level. Using Western blot methodology, we systematically examined: (1) gel composition (Tris-glycine vs. bis-Tris, gradient vs. non-gradient, sodium dodecyl sulfate (SDS) vs. no SDS); (2) sample preparation (SDS vs. no SDS, β-mercaptoethanol (BME) vs. no BME, boiling vs. no boiling); and (3) running buffer composition (SDS vs. no SDS). Our results indicate full native gel conditions prevent resolution of all collagen type 1 bands. The best resolution of type 1 procollagens is achieved using 4%–12% Tris-glycine gels without the presence of SDS in the gel itself, although SDS in the running and sample buffers are needed. Also, BME must not be added to the sample buffer and samples should not be boiled. For characterization of mature collagen 1(I), both 8% and gradients type gels are appropriate, although still without SDS, yet with SDS included in both running and sample buffers, BME must be added to the sample buffer, and samples should not be boiled. Boiling is to be avoided as the antigenic site recognized by the monoclonal antibody used is sensitive to thermal denaturation, as is the case with many monoclonal antibodies available on the market. Thus, the exact parameters employed are dependent upon the collagen protein product that the scientist desires to identify.
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答案取决于这个问题:1型肌肉胶原的蛋白质印迹表征的最佳条件取决于所需的同种型
1型纤维胶原蛋白是体内最丰富的胶原蛋白类型,是细胞外基础设施的重要组成部分。为了评估纤维化疾病中的胶原合成和细胞外积累,需要改进方法在蛋白质水平上检测前胶原和成熟胶原的变化。使用Western blot方法,我们系统地检查了:(1)凝胶组成(tris -甘氨酸vs.双tris,梯度vs.非梯度,十二烷基硫酸钠(SDS) vs.无SDS);(2)样品制备(SDS vs.无SDS, β-巯基乙醇(BME) vs.无BME,煮沸vs.不煮沸);(3)运行缓冲液成分(SDS与无SDS)。我们的结果表明,完全的天然凝胶条件阻止了所有1型胶原蛋白带的溶解。1型前胶原的最佳分辨率是使用4%-12%的tris -甘氨酸凝胶,而凝胶本身不存在SDS,尽管在运行和样品缓冲液中需要SDS。此外,BME不能添加到样品缓冲液中,也不能煮沸样品。对于成熟胶原1(I)的表征,8%和梯度型凝胶都是合适的,尽管仍然没有SDS,但是在运行缓冲液和样品缓冲液中都含有SDS,必须将BME添加到样品缓冲液中,并且样品不应煮沸。要避免煮沸,因为所用单克隆抗体识别的抗原位点对热变性很敏感,就像市场上许多单克隆抗体一样。因此,所采用的确切参数取决于科学家想要鉴定的胶原蛋白产品。
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