Wallac victor3v 1420 multilabel counter

Monomeric human α-Syn peptide at 100 µM was incubated in PBS (pH 7.4) with 25 µM thioflavin-T (ThT) in the presence or absence of various seeding agents including BSA-conjugated cyclized and linear peptide epitopes (100 nM), sonicated PFFs (10 nM), oligomers (100 nM) and physiologic tetramers (100 nM) in a 120 µL reaction volume per well of black-walled 96-well microtiter plates (Greiner Bio-One, Monroe, NC, USA). In studies of neutralizing activity, mAb was added at 0.1 nM. Plates were incubated at 37 °C with shaking for 30 sec prior to each hourly reading of ThT fluorescence (excitation at 440 nm, emission at 486 nm) using a Wallac Victor3v 1420 Multilabel Counter (PerkinElmer, Waltham, MA, USA). To be noted, in some studies, continuous shaking was used to promote aggregation, giving rise to higher levels of aggregation with monomers alone.

Monomeric Aβ42 peptide was solubilized in 10 mM NaOH to a concentration of 500 μM, sonicated for 10 min, diluted to 50 μM in 1 mM EDTA Tris-HCL buffer (pH 7.4), and immediately added to a black-walled 96-well microtitre plate (Greiner Bio-One, Monroe NC, USA). Equal volumes of buffer alone, 10 μM muPMN310 test antibody or 10 μM irrelevant mouse IgG control were added to the wells, for a final volume of 100 μL and a 1:5 molar ratio of antibody:Aβ42 peptide. Thioflavin T (ThT) was added (10 μM final) and plates were incubated at room temperature for 24 h at 25 °C, with ThT fluorescence measurements (excitation at 440 nm, emission at 486 nm) recorded every hour using a Wallac Victor3v 1420 Multilabel Counter (PerkinElmer, Waltham MA, USA). Fluorescent readings were double-referenced by buffer alone and antibody only wells.
At end-point, samples were collected and the wells washed with 10 mM NaOH. The collected samples were centrifuged at 18,000 g for 14 min at 4 °C, and pellets were resuspended in 10 mM NaOH. After solubilization in sample buffer and boiling for 5 min at 95 °C, supernatants and pellets were run on a denaturing SDS-PAGE gel for monomerization and immunoblotting with a pan-Aβ antibody (6E10, BioLegend, San Diego CA, USA). Total Aβ in the pellet or supernatant appears as an approximately 4 kDa band.

For direct binding measurements by fluorescence polarization, increasing amounts of the indicated protein were added to a 50 nM solution of a 5′ fluorescein-labeled synthetic 145 bp duplex DNA (Widom-601 sequence^{68 (link)}; 5′-ATC AGA ATC CCG GTG CCG AGG CCG CTC AAT TGG TCG TAG ACA GCT CTA GCA CCG CTT AAA CGC ACG TAC GCG CTG TCC CCC GCG TTT TAA CCG CCA AGG GGA TTA CTC CCT AGT CTC CAG GCA CGT GTC AGA TAT ATA CAT CGA T-3′) in 20 mM Tris pH 7.5, 150 mM NaCl, 1% glycerol, 0.5 mg mL⁻¹ BSA and 0.05% Triton X-100. After 15 min incubation at room temperature, samples (20 μL) were placed in a black 384-well polypropylene plate (Greiner Bio-One) and fluorescence polarization was measured using a Wallac Victor3V 1420 Multilabel Counter (PerkinElmer). Competition assays were performed using a fixed concentration of BAZ1A-PHD (5.3 μM; apparent K_D) and adding increasing amounts of unlabeled synthetic duplex 145 bp DNA with identical sequence to the fluorescein-labeled probe. K_D and IC₅₀ values and standard errors were determined by fitting the data from three independent experiments to a four-parameter binding model using Prism 6 (version 6.0e, GraphPad).