Alpha acceptor beads

The following item was purchased from ATCC: Vero-E6 (CRL-1586, RRID:CVCL_0574). The following items were purchased from Corning TM: EMEM (10–009-CV), HI FBS (35–016-VC) and 0.25% Trypsin (25053CI). The untagged NP (Z03501) was purchased from Genscript. The His-tagged NP (40588-V08B) was purchased from SinoBiological.
The following item was purchased from Gibco: Pen/Strep (15140–122). PBS (SH30256FS) was purchased from HyClone. The following items were purchased from PerkinElmer: ProxiPlate-384 Plus (Cat# 6008280), CulturPlate-384 (Cat#: 6007680), Alpha Streptavidin Donor beads (6760002), AlphaLISA lysis buffer (AL003C). The following custom labeling was performed by PerkinElmer: donor antibodies were biotinylated using NHS activated biotinylating reagent (ChromaLink #B-1001–105), and acceptor antibodies were conjugated to Alpha Acceptor Beads (PerkinElmer #6760137M).

Synthesized protein-protein interactions were assessed using the amplified luminescent proximity homogeneous assay (ALPHA). A total of 100 ng of each protein was added to ALPHA buffer [100 mM Tris-HCl (pH 8.0), 0.01% (v/v) Tween20], 1 mg/mL of BSA, 17 μg/mL of streptavidin-conjugated ALPHA donor beads (PerkinElmer, Waltham, MA, USA), 17 μg/mL of protein-A-conjugated ALPHA acceptor beads, and 5 μg/mL of anti-FLAG mAb M2 and incubated in a 1/2ALPHAPlate-96 shallow well (PerkinElmer, Waltham, MA, USA) at 25 °C for 24 h. The fluorescence emission signals of each well were measured using an EnSpire™ Multimode Plate Reader (PerkinElmer, Waltham, MA, USA).
The interaction between NLRP3 and Aβ was assessed by incubating 100 ng of NLRP3-FL-Btn with 5 μg/mL of anti-Aβ mAb (6E10) (BioLegend, San Diego, CA, USA), 17 μg/mL of protein-A-conjugated ALPHA acceptor beads, and 17 μg/mL of streptavidin-conjugated ALPHA donor beads for 24 h with the indicated concentrations of Aβ and incubated in a 1/2ALPHAPlate-96 shallow well (PerkinElmer, Waltham, MA, USA) at 25 °C for 24 h. The fluorescence emission signals of each well were measured using an EnSpire™ Multimode Plate Reader (PerkinElmer, Waltham, MA, USA).