Zeba spin desalting columns 7k mwco 0.5 ml

Rat IgG (ThermoFisher catalog #31933) was labeled with I-125 via Pierce Iodination Beads (ThermoFisher catalog # 28665). We then conjugated the IgG to nanoparticles using our published protocol[15 (link)]. In brief, 100 μL of polystyrene NPs were buffer exchanged with Zeba Spin Desalting Columns, 7K MWCO, 0.5 mL (ThermoFisher catalog # 89882), exchanging for 50 mM MES buffer at pH 5.2, finally putting the buffer-exchanged beads into 1.5 mL Eppendorf tubes. Next N-Hydroxysulfosuccinimide (“sulfo-NHS”; Sigma catalog #56485) was added to a final concentration of 0.275 mg/ml and incubated for 3 minutes at room temperature (RT). Next N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (“EDAC”; Sigma catalog # E7750) was added to a final concentration of 0.1 mg/ml and incubated for 10–15 minutes at RT. Next 114 ug of I-125-labeled rat IgG was added (giving 200 antibody molecules per bead) and incubated for 2 to 4 hours at RT on a vortex/shaker at low speed. 1 mL MES buffer was added to dilute free antibody, followed by centrifuge at 12,000g x 3 min to pellet the IgG-conjugated NPs. The IgG-NP pellet was resuspended in 200 uL of PBS + 0.05% bovine serum albumin (BSA) buffer. Immediately before use, the NPs were sonicated with a probe / tip sonicator (Qsonica Q55 Sonicator, Cole-Parmer #UX-04712-51) for three 3-second pulses at 30% maximum power.

FTO activity was determined using the FTO Chemiluminescent Assay Kit (BPS Bioscience). Before reaction, FTO samples purified from either E. coli or BES were incubated with 0.5 mM EDTA for 5 min and then underwent one of two procedures: buffer exchange on a salting-out column (Zeba™ Spin Desalting Columns, 7K MWCO, 0.5 mL, Thermo Scientific™, Waltham, MA, USA, #89882) or overnight dialysis. The measurements were carried out according to the manufacturer’s protocol with RNA containing N⁶-meA (provided in the kit) as a substrate. Chemiluminescence was measured using the Synergy HT (Bio Tek, Winooski, USA) plate reader with an integration time of 1 s and 0.1 s delay after the plate movement. The demethylase activity of a given FTO sample was estimated according to the following formula, and further adjusted for protein concentration.

where S_sample and S_bkg are the chemiluminescence intensities for reaction containing a protein sample and the buffer background, respectively, and S_ECFTO is the mean signal intensity by a reference sample containing 1 µg of purified ^ECFTO and determined for 2–3 independent preparations.

For the selection of hSIRPα-specific Nbs, two consecutive phage enrichment rounds either with immobilized hSIRPα or hSIRPαD1 were performed. To generate Nb-presenting phages, TG1 cells comprising the Nb-library in pHEN4 were infected with the M13K07 helper phage. In each panning round, 1 × 10¹¹ phages were applied to streptavidin or neutravidin plates (Thermo Fisher Scientific) coated with biotinylated antigen (5 µg/mL). For biotinylation, purified antigen (Acrobiosystems) was reacted with Sulfo-NHS-LC-LC-Biotin (Thermo Fisher Scientific) in 5 M excess at ambient temperature for 30 min. Excess of biotin was removed by size exclusion chromatography using Zeba™ Spin Desalting Columns 7K MWCO 0.5 mL (Thermo Fisher Scientific) according to the manufacturer’s protocol. Blocking of antigen and phage was performed alternatively with 5% milk or Bovine Serum Albumin (BSA) in Phosphate-Buffered Saline with Tween (PBS-T), and, as the number of panning rounds increased, the wash stringency with PBS-T was intensified. Bound phages were eluted in 100 mM triethylamine (TEA) (pH 10.0), followed by immediate neutralization with 1 M Tris/HCl (pH 7.4). Exponentially growing TG1 cells were infected with eluted phages and spread on selection plates for subsequent selection rounds. In each round, antigen-specific enrichment was monitored by counting colony-forming units.

The antibodies, mAb3D6 and mAb3D6-scFv8D3, were labeled with iodine-125 (¹²⁵I) by direct iodination with chloramine T [36 (link)]. Briefly, antibody (mAb3D6 or mAb3D6-scFv8D3), ¹²⁵I stock solution (Perkin Elmer, USA) and chloramine T (5 µg) were mixed in PBS to a final volume of 110 μL, and then incubated 90 s in room temperature. The labeling reaction was quenched with 10 μg sodium metabisulfite. Radioiodinated antibody was purified with Zeba Spin Desalting Columns (7 K MWCO, 0.5 mL, Thermo Fisher Scientific, Waltham, MA, USA). Binding of [¹²⁵I]I-mAb3D6 and [¹²⁵I]I-mAb3D6-scFv8D3 to Aβ and TfR was tested with ELISA directly after radiolabeling according to a previously described method [16 (link)].