School of Biological Sciences, Georgia Institute of Technology; Department of Structural Biology, Genentech.
School of Biological Sciences, Georgia Institute of Technology.
J Vis Exp. 2022 Jun 29(184). doi: 10.3791/64099.
The peel-blot cryo-EM grid preparation technique is a significantly modified back-injection method with the objective of achieving a reduction in layers of multi-layered samples. This removal of layers prior to plunge freezing can aid in reducing sample thickness to a level suitable for cryo-EM data collection, improving sample flatness, and facilitating image processing. The peel-blot technique allows for the separation of multilamellar membranes into single layers, of layered 2D crystals into individual crystals, and of stacked, sheet-like structures of soluble proteins to likewise be separated into single layers. The high sample thickness of these types of samples frequently poses insurmountable problems for cryo-EM data collection and cryo-EM image processing, especially when the microscope stage must be tilted for data collection. Furthermore, grids of high concentrations of any of these samples can be prepared for efficient data collection since sample concentration prior to grid preparation can be increased and the peel-blot technique adjusted to result in a dense distribution of single-layered specimen.
膜片-印迹冷冻电镜制样技术是一种经过显著改良的反向注射方法，其目的是减少多层样品的层数。在倾入式快速冷冻之前去除这些层，可以帮助将样品厚度降低到适合冷冻电镜数据采集的水平，提高样品平整度，并促进图像处理。膜片-印迹技术可将多层膜分离成单层、层状 2D 晶体分离成单个晶体，以及将可溶性蛋白的堆叠片状结构分离成单层。这些类型的样品的高样品厚度通常给冷冻电镜数据采集和冷冻电镜图像处理带来难以克服的问题，特别是在显微镜台必须倾斜以进行数据采集时。此外，由于可以增加网格制备前的样品浓度，并调整膜片-印迹技术以实现单层标本的密集分布，因此可以制备高浓度的任何这些样品的网格，以实现高效的数据采集。
