A practical guide to the use of monolayer purification and affinity grids.
Lipid monolayers have traditionally been used in electron microscopy (EM) to form two-dimensional (2D) protein arrays for structural studies by electron crystallography. More recently, monolayers containing Nickel-nitrilotriacetic acid (Ni-NTA) lipids have been used to combine the purification and preparation of single-particle EM specimens of His-tagged proteins into a single, convenient step. This monolayer purification technique was further simplified by introducing the Affinity Grid, an EM grid that features a predeposited Ni-NTA lipid-containing monolayer. In this contribution, we provide a detailed description for the use of monolayer purification and Affinity Grids, discuss their advantages and limitations, and present examples to illustrate specific applications of the methods.
Kelly
Deborah F
DF
Department of Cell Biology, Harvard Medical School, Boston, Massachusetts, USA.
Dukovski
Danijela
D
Walz
Thomas
T
D020285:Cryoelectron Microscopy / Q000379:methods*; D003461:Crystallography / Q000379:methods*; D008055:Lipids / Q000737:chemistry*; D009532:Nickel / Q000737:chemistry; D009571:Nitrilotriacetic Acid / Q000737:chemistry; D011506:Proteins / Q000737:chemistry / Q000648:ultrastructure
D008055:Lipids; D011506:Proteins; D009532:Nickel; D009571:Nitrilotriacetic Acid