BUILDING UP THE FACILITY FOR MEMBRANE PROTEIN MANIPULATION AND SPIN LABELING

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 ACERT的主要活动之一是开发技术，以增加生物医学研究的产量。这一重要活动将加强ACERT的核心研究和合作，但也将通过开发更灵敏、更有成效的脉冲ESR光谱仪，使其服务和传播活动受益。鉴于提高灵敏度具有最高的优先事项，不应在样品制备部分将这项任务与PDS光谱学的另一面分开考虑，因为良好的样品质量也是实现高通量的关键。为了实现这一目标，我们正在建立一个小型但设备齐全的本地设施，适合各种蛋白质生化操作。重点将放在膜蛋白的研究上，日常活动将涉及到有效的蛋白质自旋标记；重组为脂质体、纳米盘或双胞体、透析、样品浓缩等。我们希望通过能够制备和研究新鲜的膜蛋白样本来提高工作质量，因为它们通常非常精细，不能容忍过度的操作，例如冷冻/解冻循环。它将使开发最有效的自旋标记协议，取决于特定的蛋白质，从而帮助我们的合作者谁是有限的或缺乏能力来测试自旋标记和优化样本的PDS研究。ACERT的工作人员将提供ACERT用户在ESR谱学样品制备方面的支持和培训，特别是脉冲偶极ESR样品制备。我们相信，这样的设施将有助于我们的生物医学研究的灵活性和优点，并将支持该中心的效率。此外，这样的设施将使ACERT的科学家能够通过提供现场访问标准技术，如荧光光谱学和圆二色谱，常规地应用补充ESR研究的方法。这将加强我们现有的研究，并使我们能够快速实施新的想法，这将有利于合作项目、核心研究和服务活动。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. One of the major activities at ACERT is aimed at developing technologies for increasing the throughput of biomedical research. This important activity is poised to strengthen ACERT core research and collaborations, but also to benefit its service and dissemination activates by means of developing more sensitive and thereby considerably more productive pulse ESR spectrometers. Given that increasing sensitivity carries the highest priority, this task should not be considered in isolation from the other side of PDS spectroscopy in the part of sample preparation, since sample good quality is also essential for achieving high throughput. To address this goal, we are setting up a small but well-equipped local facility, suited for variety of biochemical manipulation with proteins. The emphasis will be given to the research on membrane proteins and everyday activities will be addressing efficient protein spin labeling; reconstitutions into liposomes, nanodisks or bicelles, dialysis, sample concentration, etc. We expect to improve the quality of our work by having been able to prepare and study fresh membrane protein samples, since they often are very delicate and do not tolerate excessive manipulations, for example freezing/thawing cycles. It will enable developing the most efficient protocols for spin-labeling, depending on the particular protein, thus helping our collaborators who have limited or lacking capacity to assay spin-labeling and optimize samples for PDS study. ACERT user support and training in sample preparation for ESR spectroscopy, in particular for pulsed dipolar ESR will be provided by ACERT staff. We are confident that such facility will contribute to the flexibility and merits of our biomedical research and will back the efficiency of the center. Additionally, such a facility will enable ACERT scientists to apply routinely the methods complementary to the ESR studies by providing on site access to standard techniques, such as fluorescent spectroscopy and circular dichroism. This will strengthen our existing research, and enable us to quickly implement the new ideas, which will be beneficial for collaborative projects, core research, and service activities.