Extended Data Fig. 1: Biochemical characterization of native receptor isolation from mouse brains using an engineered Fab fragment.
From: Cryo-EM structures reveal native GABAA receptor assemblies and pharmacology
a and b, Expression of tri-heteromeric GABAARs with different α subunits and binding test with the engineered 8E3-GFP Fab monitored with fluorescence-detection size-exclusion chromatography (FSEC). The signal is from the fusion-red protein inserted into the intracellular loop of the γ2 subunit in panel a or the GFP of the 8E3-GFP Fab in panel b. c and d, FSEC traces demonstrating the superior capturing efficiency and protein yield of streptactin-XT resin. e, Western blot analysis of steps during the native receptor purification. The neuroligin 2 (NL2) immunoblot (Synaptic Systems 129 202, 1:1000 dilution) shows robust solubilization of the inhibitory synapse marker NL2. Lanes from left to right were the membrane input and the LMNG solubilized supernatant. The α1 subunit immunoblot (Millipore 06–868, 1:1000 dilution) shows quantitation of the α1 subunit during membrane solubilization, affinity capturing, and elution steps. The numbers on green lines are trimmed signal from the 800 nm channel (shown as green) used for quantitation. The numbers on red lines are trimmed signal from the 700 nm channel (shown in red), which should be close to zero. Western blot detection of the NL2 and the quantitative analysis of the α1 subunit were repeated twice with comparable results. f, The workflow of nα1-GABAARs purification from mouse brains. g, Size-exclusion chromatography (SEC) of nα1-GABAARs and silver-stain SDS-PAGE analysis of different SEC fractions. The SEC and SDS-PAGE analysis on nα1-GABAARs were repeated more than 5 times with similar peak profile and band pattern. h, Identification of GABAAR subunits from the pentameric peak using mass spectrometry. #PSM indicates the number of peptide spectrum matches, coverage refers to the sequence coverage of protein subunits from identified peptides, and ND indicates ‘not detected’. i, Negative-staining electron microscopy images of protein samples from the pentameric peak. j, Scintillation proximity assay of the pentameric peak fraction with 3H-flunitrazepam, at each concentration the specific count is shown as mean ± s.d. (n = 3 replicates prepared from 1 independent native receptor preparation).
