| Title: |
Streptavidin-Affinity Grid Fabrication for Cryo-Electron Microscopy Sample Preparation. |
| Authors: |
Cookis, Trinity; Sauer, Paul; Poepsel, Simon; Han, Bong-Gyoon; Herbst, Dominik A; Glaeser, Robert; Nogales, Eva |
| Source: |
Journal of Visualized Experiments, iss 202 |
| Publisher Information: |
eScholarship, University of California |
| Publication Year: |
2023 |
| Collection: |
University of California: eScholarship |
| Subject Terms: |
3101 Biochemistry and Cell Biology (for-2020); 31 Biological Sciences (for-2020); Generic health relevance (hrcs-hc); Cryoelectron Microscopy (mesh); Streptavidin (mesh); Carbon (mesh); Biotin (mesh); Water (mesh); 0601 Biochemistry and Cell Biology (for); 1701 Psychology (for); 1702 Cognitive Sciences (for) |
| Description: |
Streptavidin affinity grids provide strategies to overcome many commonly encountered cryo-electron microscopy (cryo-EM) sample preparation challenges, including sample denaturation and preferential orientations that can occur due to the air-water interface. Streptavidin affinity grids, however, are currently utilized by few cryo-EM labs because they are not commercially available and require a careful fabrication process. Two-dimensional streptavidin crystals are grown onto a biotinylated lipid monolayer that is applied directly to standard holey-carbon cryo-EM grids. The high-affinity interaction between streptavidin and biotin allows for the subsequent binding of biotinylated samples that are protected from the air-water interface during cryo-EM sample preparation. Additionally, these grids provide a strategy for concentrating samples available in limited quantities and purifying protein complexes of interest directly on the grids. Here, a step-by-step, optimized protocol is provided for the robust fabrication of streptavidin affinity grids for use in cryo-EM and negative-stain experiments. Additionally, a trouble-shooting guide is included for commonly experienced challenges to make the use of streptavidin affinity grids more accessible to the larger cryo-EM community. |
| Document Type: |
article in journal/newspaper |
| File Description: |
application/pdf |
| Language: |
unknown |
| Relation: |
qt19c9j0sp; https://escholarship.org/uc/item/19c9j0sp; https://escholarship.org/content/qt19c9j0sp/qt19c9j0sp.pdf |
| DOI: |
10.3791/66197 |
| Availability: |
https://escholarship.org/uc/item/19c9j0sp; https://escholarship.org/content/qt19c9j0sp/qt19c9j0sp.pdf; https://doi.org/10.3791/66197 |
| Rights: |
public |
| Accession Number: |
edsbas.50DCDB7C |
| Database: |
BASE |