Chen Shanyan, Meng Fanjun, Chen Zhenzhou, Qu Zhe, Cui Jiankun, Gu Zezong
Department of Pathology and Anatomical Sciences, University of Missouri School of Medicine, Columbia, MO, USA.
Interdisciplinary Neuroscience Program, University of Missouri, Columbia, MO, USA.
Methods Mol Biol. 2017;1626:147-155. doi: 10.1007/978-1-4939-7111-4_13.
Pathological activation of gelatinases (matrix metalloproteinase-2 and -9; MMP-2/-9) has been shown to cause a number of detrimental outcomes in neurodegenerative diseases. In gel gelatin zymography is a highly sensitive methodology commonly used in revealing levels of gelatinase activity and in separating the proform and active form of gelatinases, based on their different molecular weights. However, this methodology is inadequate in resolving complex enzyme isoforms, because gelatinase expression and activity can be regulated at transcriptional and/or post-translational levels under in vivo conditions resulting in alternation of their isoelectric focusing (IEF) points. In this chapter, we describe an advanced methodology, termed two-dimensional zymography, combining IEF with zymographic electrophoresis under non-reducing conditions to achieve significant improvement in separation of the gelatinase isoforms in both cell-based and in vivo models for acute brain injuries and neuroinflammation.
明胶酶（基质金属蛋白酶-2和-9；MMP-2/-9）的病理性激活已被证明会在神经退行性疾病中导致许多有害后果。在凝胶中，明胶酶谱法是一种高度灵敏的方法，常用于揭示明胶酶活性水平，并根据其不同分子量分离明胶酶的前体形式和活性形式。然而，这种方法在解析复杂的酶同工型方面存在不足，因为在体内条件下，明胶酶的表达和活性可在转录和/或翻译后水平受到调控，从而导致其等电聚焦（IEF）点发生改变。在本章中，我们描述了一种先进的方法，称为二维酶谱法，即在非还原条件下将IEF与酶谱电泳相结合，以显著改善在急性脑损伤和神经炎症的基于细胞和体内模型中明胶酶同工型的分离。
