Amicon ultra 0.5 ml centrifugal filter device

Folded origami nanostructures were purified from excess staples by poly(ethylene) glycol (PEG) precipitation (Stahl et al., 2014 (link)). Unpurified origami structures were mixed 1:1 with PEG precipitation buffer (TE buffer, 12.5 mM MgCl₂, 500 mM NaCl, 15% w/v PEG 8000) and pelleted by centrifugation at 20,000 x g for 30 min at 20°C. The origami pellet was then resuspended in 100 μL folding buffer (TE buffer with 12.5 mM MgCl₂). The PEG precipitation step is repeated two more times, and the pellet is resuspended in Folding Buffer to the desired concentration.
Excess PEG may be removed by molecular weight cut-off (MWCO) filtration. For MWCO purification, the PEG-purified sample is supplemented with Folding Buffer to a final volume of 500 μL, applied to a 100 kDa Amicon Ultra-0.5 mL centrifugal filter device (Merck), and concentrated by centrifugation at 14,000 x g for 15 min at 20°C. 3 centrifugation steps are applied before recovering the sample, and the mixture is supplemented with 485 μL Folding buffer before each successive step. After centrifugal removal of PEG, initial volumes of 100 μL and 1 mL yield around 20 μL of origami at 100-200 nM and 1-2 μΜ respectively. For cryoET, concentrations of ∼1 μM are typically desired.

HSP-specific monoclonal antibodies (SPA-810 and SPA-800 for HSP70 and HSP27, respectively) were conjugated to Homebrew carboxylated paramagnetic beads (Quanterix Corporation, Cambridge, MA, USA) following a standard coupling chemistry, based on 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide, according to the manufacturer’s protocol (Quanterix Corporation, Cambridge, MA, USA). Briefly, the monoclonal antibodies were buffer exchanged into 50 mM 2-(N-morpholino) ethanesulfonic acid (bead conjugate buffer, Quanterix Corporation, Cambridge, MA, USA) using Amicon^® Ultra 0.5-mL centrifugal filter devices (EMD Millipore, Billerica, MA, USA). After that, the beads were washed three times with PBS containing 1% Tween 20 (PBS/T) and twice with bead conjugate buffer, pH 6.2. Then, the beads were activated with 0.5 mg/mL freshly prepared 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide in cold bead conjugate buffer for 30 min. The activated beads were then washed with cold bead conjugate buffer and conjugated with appropriate concentration of HSP-specific antibodies in bead conjugate buffer. After 2 h, the capture beads were washed twice with PBS/T and blocked with PBS containing 1% bovine serum albumin (PBS/BSA) for 30 min. Finally, the coated and blocked beads were washed once with PBS/T, then with 50 mM Tris buffer supplemented with 1% BSA and stored in the latter at 4 °C until use.