Chapso

For assay optimization phosphate-, tris(hydroxymethyl)aminomethan (TRIS)- and triethanolamine (TEA)-based buffers with different concentrations of dimethyl sulfoxide (DMSO) were compared. Furthermore, different reporter molecules, reagent concentrations, detection systems, and the use of detergents (Tween-20, Triton X-100, Triton X-114, IGEPAL, Brij 35, CHAPSO; Sigma-Aldrich) were evaluated. Following these multiple optimization steps, a new standard protocol was defined: MaxiSorp immunoassay plates were coated with 5 μg/ml of mAb JD5.1 overnight at 4°C. After washing the plate twice with PBST, the plate was blocked with SuperBlock T20 (TBS) blocking buffer (Thermo Scientific) for 1 h at 37°C. After another washing step, serial dilutions of the samples in LW buffer (0.2 M TEA, pH 7.5, with 20% DMSO) were added to the plates and incubated in the dark for 2 h at 37°C. Without washing, 100 μL of an 80 ng/ml solution of the reporter molecule MG-161 (Fig 1) in LW buffer was added to the plate and incubated for an additional 45 min. Subsequently, plates were washed four times, and bound MG-161 was detected after 1 h incubation at 37°C by horseradish peroxidase-coupled streptavidin (SouthernBiotech) diluted 1:5000 in PBST. Signal development was done with 3,3’,5,5’-tetramethylbenzidine (TMB) for 5 min after which the reaction was stopped with 0.5 M sulphuric acid.

Proteins were concentrated using a centricon (Amicon) with a 30-kDa cutoff and buffer exchanged into 20 mM Hepes (pH 7.6), 200 mM NaCl, 0.5 mM EDTA, 2 mM MgCl₂, and 5 mM DTT. Purified Ku70/80 full length was then first mixed with Y-shaped 42- to 55-bp DNA before being mixed with purified DNA-PKcs, LX4, and PAXX in a 2:2:2:2 ratio respectively.
Aliquots of 3 μl of the NHEJ super complex (~2.5 mg/ml) were mixed with 8 mM CHAPSO to eliminate particle orientation bias (final concentration; Sigma-Aldrich) before being applied to Holey carbon grids (Quantifoil Cu R1.2/1.3, 300 mesh), glow-discharged for 60 s at a current of 25 mA in PELCO easiGlow (Ted Pella Inc.). The grids were then blotted with filter paper once to remove any excess sample and plunge-frozen in liquid ethane using a FEI Vitrobot Mark IV (Thermo Fisher Scientific) at 4°C and 95% humidity.

A standard liposome leakage assay contained 40 μL of reconstitution buffer without glycerol, 5 μL of sulforhodamine B-loaded liposomes, and 5 μL of the desired concentration of JD1. The leakage assay was conducted in a 96-well microplate at 37°C, and sulforhodamine B fluorescence (ex565/em585 nm) was measured every 2 minutes in a BioTek Synergy H1 plate reader. Ten μL of 10% CHAPSO (Sigma-Aldrich) was added to each sample at the end of the reaction [123 (link)]. Leakage data were presented as the percentage of maximum fluorescence change based on three biological replicates.

CHAPSO detergent resistant membranes (DRMs) were prepared from brain cortices after careful removal of leptomeninges and blood vessels, as previously described (Szaruga et al., 2015 (link)). Briefly, tissue was homogenized in ∼10 volumes of 10% sucrose in MBS buffer (25 mM MES, pH 6.5, 150 mM NaCl) containing 1% CHAPSO (Sigma); separated by a sucrose density gradient and the DRM fraction (interface of 5%/35% sucrose) was collected and rinsed twice in 20 mM PIPES, pH 7, 250 mM sucrose, 1 M EGTA. The resultant pellet was resuspended with the above buffer and used as source of enzyme. Activity assays were carried out for 1 or 2 h for mouse or human derived DRMs, respectively, as described before (Szaruga et al., 2015 (link)).