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Streptavidin-Affinity Grid Fabrication for Cryo-Electron Microscopy Sample Preparation.
- Source :
-
Journal of visualized experiments : JoVE [J Vis Exp] 2023 Dec 29 (202). Date of Electronic Publication: 2023 Dec 29. - Publication Year :
- 2023
-
Abstract
- 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.
- Subjects :
- Cryoelectron Microscopy methods
Streptavidin chemistry
Water
Carbon chemistry
Biotin
Subjects
Details
- Language :
- English
- ISSN :
- 1940-087X
- Issue :
- 202
- Database :
- MEDLINE
- Journal :
- Journal of visualized experiments : JoVE
- Publication Type :
- Academic Journal
- Accession number :
- 38224121
- Full Text :
- https://doi.org/10.3791/66197