分离、鉴定和验证葡萄糖或碳水化合物结合受体的方案。

IF 2.5 Q3 BIOCHEMICAL RESEARCH METHODS Biology Methods and Protocols Pub Date : 2024-06-19 eCollection Date: 2024-01-01 DOI:10.1093/biomethods/bpae045
Nadia Rashid, Kavaljit H Chhabra
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引用次数: 0

摘要

葡萄糖的感知、运输和利用对于维持动物体内的能量平衡至关重要。尽管参与葡萄糖在不同细胞间隙中移动的转运体已相当为人所知,但与葡萄糖结合以独立于葡萄糖代谢介导其效应的受体在很大程度上仍未被认识。建立精确且可重复的方法来鉴定大脑或其他外周器官中的葡萄糖受体,将为理解葡萄糖信号通路在维持、调节和重塑细胞代谢需求中的作用铺平道路。鉴定这些潜在的葡萄糖受体还可能开发出治疗糖尿病和相关代谢紊乱的有效疗法。市售的生物素或放射性标记葡萄糖共轭物分子量较低,因此无法提供足够的灵敏度和密度来分离葡萄糖受体。在此,我们介绍一种利用高分子量葡萄糖(或其他碳水化合物)共轭物分离、鉴定和验证葡萄糖结合受体的方法。我们已制备出 30 kDa 葡萄糖(或其他碳水化合物)生物素-聚丙烯酰胺(PAA)共轭物,其摩尔分数分别为 80:5:15%。这些共轭物可与生物素-链霉亲和素生物化学、细胞内酶联免疫吸附和表面等离子体共振(SPR)方法一起用于分离、鉴定和验证葡萄糖或碳水化合物结合受体。我们首先展示了如何利用链霉亲和素涂层磁珠固定葡萄糖-生物素-PAA 共轭物。然后,利用这些磁珠从组织匀浆或单细胞悬浮液中富集和分离葡萄糖结合蛋白。对富集或分离的蛋白质进行质谱/蛋白质组学分析,以揭示作为潜在葡萄糖受体的顶级候选蛋白质的身份。然后,我们将介绍如何利用细胞内 ELISA 方法,通过受体在体外的稳定表达来验证葡萄糖与其潜在受体之间的相互作用。我们进一步展示了如何使用高灵敏度的 SPR 方法来测量葡萄糖与其受体的结合动力学。总之,我们介绍了一种利用磁珠、细胞内 ELISA 和 SPR 分离、鉴定和验证葡萄糖或碳水化合物结合受体的方法。该方案将成为未来研究健康和疾病中葡萄糖或碳水化合物受体信号通路的基础。
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A protocol to isolate, identify, and verify glucose- or carbohydrate-binding receptors.

Sensing, transport, and utilization of glucose is pivotal to the maintenance of energy homeostasis in animals. Although transporters involved in mobilizing glucose across different cellular compartments are fairly well known, the receptors that bind glucose to mediate its effects independently of glucose metabolism remain largely unrecognized. Establishing precise and reproducible methods to identify glucose receptors in the brain or other peripheral organs will pave the way for comprehending the role of glucose signaling pathways in maintaining, regulating, and reprogramming cellular metabolic needs. Identification of such potential glucose receptors will also likely lead to development of effective therapeutics for treatment of diabetes and related metabolic disorders. Commercially available biotin or radiolabeled glucose conjugates have low molecular weight; therefore, they do not provide enough sensitivity and density to isolate glucose receptors. Here, we describe a protocol to isolate, identify, and verify glucose-binding receptor/s using high molecular weight glucose (or other carbohydrate) conjugates. We have produced 30 kDa glucose- (or other carbohydrate-) biotin-polyacrylamide (PAA) conjugates with mole fractions of 80:5:15% respectively. These conjugates are used with biotin-streptavidin biochemistry, In-cell ELISA, and surface plasmon resonance (SPR) methods to isolate, identify, and verify glucose- or carbohydrate-binding receptors. We first demonstrate how streptavidin-coated magnetic beads are employed to immobilize glucose-biotin-PAA conjugates. Then, these beads are used to enrich and isolate glucose-binding proteins from tissue homogenates or from single-cell suspensions. The enriched or isolated proteins are subjected to mass spectrometry/proteomics to reveal the identity of top candidate proteins as potential glucose receptors. We then describe how the In-cell ELISA method is used to verify the interaction of glucose with its potential receptor through stable expression of the receptor in-vitro. We further demonstrate how a highly sensitive SPR method can be used to measure the binding kinetics of glucose with its receptor. In summary, we describe a protocol to isolate, identify, and verify glucose- or carbohydrate-binding receptors using magnetic beads, In-cell ELISA, and SPR. This protocol will form the future basis of studying glucose or carbohydrate receptor signaling pathways in health and in disease.

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来源期刊
Biology Methods and Protocols
Biology Methods and Protocols Agricultural and Biological Sciences-Agricultural and Biological Sciences (all)
CiteScore
3.80
自引率
2.80%
发文量
28
审稿时长
19 weeks
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