| Expression and Purification of Yeast-derived GPCR, Gα and Gβγ Subunits for Structural and Dynamic Studies
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Author:
Date:
2021-02-20
[Abstract] In the last several years, as evidence of a surged number of GPCR-G complex structures, the expressions of GPCRs and G proteins for structural biology have achieved tremendous successes, mostly in insect and mammalian cell systems, resulting in more than 370 structures of over 70 GPCRs have been resolved. However, the challenge remains, particularly in the conformational transition and dynamics study area where a much higher quantity of the receptors and G proteins is required even in comparison to X-ray and cryo-EM (5 mg/ml, 3 μl/sample) when NMR spectroscopy (5 mg/ml, 250 μl /sample) ...
[摘要] [摘要]在过去的几年中，作为GPCR-G复杂结构数量激增的证据，用于结构生物学的GPCR和G蛋白的表达已取得了巨大的成功，主要是在昆虫和哺乳动物细胞系统中，导致了370多个已解决了70多个GPCR的结构。但是，挑战仍然存在，特别是在构象转变和动力学研究领域，即使与X射线和冷冻EM相比（5 mg / ml，3μl /样品），也需要大量的受体和G蛋白。当应用NMR光谱法（5 mg / ml，250μl /样品）时。结果，i的表达水平 nsect和哺乳动物系统也很难满足这一需求，更不用说使用绝大多数系统使用这些系统生产GPCR和G蛋白的成本高昂了。因此，需要探索一种具有广泛适用性的有效，负担得起的实用方法。毕赤酵母表达系统已在GPCR制备中显示出其希望，并具有其他真核表达系统无法比拟的许多优点。在该系统中表达的GPCR价格便宜，易于操作，并且能够进行同位素标记。在此，我们提出最近开发并在我们的实验室升级的相关协议，包括表达和纯化的毕赤酵母衍生GPCR以G沿α和G βγ蛋白。我们预期这些协议将促进GPCR及其复合物的构象转变和动力学研究。
[背景] G蛋白偶联受体（GPCR）是最大的膜蛋白家族，在许多（病理）生理活动中起着关键作用。GPCR的或它们的效应物的功能障碍会导致各种病症，包括神经变性疾病，癌症，和慢性炎症（Overington等人，2006） ...
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| Detection of Tumor Cell Surface-reactive Antibodies
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Author:
Date:
2014-05-05
[Abstract] Vaccine-based immunotherapy is being used to treat dogs with primary brain tumors. The vaccines are composed of a lysate of autologous tumor cells, which stimulate an immune response producing tumor specific antibodies that are capable of inducing antibody-dependent cell-mediated cytotoxicity to allogeneic, as well as autologous, tumor cells. This protocol will describe the tumor cell serum antibody-binding assay to measure the tumor-reactive IgG antibody response. Key features of this assay are that it is performed with sera collected from the canine patient prior to and following ...
[摘要] 基于疫苗的免疫疗法被用于治疗具有原发性脑肿瘤的狗。 疫苗由自体肿瘤细胞的裂解物组成，其刺激产生免疫应答的肿瘤特异性抗体，其能够诱导对同种异体以及自体的肿瘤细胞的抗体依赖性细胞介导的细胞毒性。 该方案将描述肿瘤细胞血清抗体结合测定以测量肿瘤反应性IgG抗体应答。 该测定的主要特征是，在接种疫苗之前和之后从犬患者收集的血清作为抗体的来源进行，并且使用犬脑肿瘤细胞作为靶细胞。
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| Natural Killer Cell Transfer Assay
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Author:
Date:
2013-08-20
[Abstract] Natural Killer (NK) cells are cytotoxic lymphocytes that constitute a major component of the innate immune system. Immunosurveillance of the host by NK cells for malignant and virally-infected cells results in direct cytotoxicity and the production of cytokines to enhance the immune response. This protocol will describe the adoptive transfer of purified NK cells into NK cell-deficient tumor bearing mice in order to establish the intrinsic functionality of NK cells.
[摘要] 天然杀伤（NK）细胞是细胞毒性淋巴细胞，其构成先天性免疫系统的主要组分。 通过NK细胞对恶性和病毒感染的细胞的宿主的免疫监视导致直接的细胞毒性和细胞因子的产生，以增强免疫应答。 该方案将描述纯化的NK细胞过继转移到NK细胞缺陷的荷瘤小鼠中以建立NK细胞的内在功能。
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