Glomax explorer plate reader

The reaction mix for Cas13 activity was prepared by combining 8.6 μl of nuclease-free water, 2 μl of cleavage buffer (400 mM Tris pH 7.4), 2 μl of LwaCas13a protein diluted in Storage Buffer (SB) to a concentration of 126.6 μg/ml, 1 μl of crRNA (40 ng/μl), 1 μl of the fluorescent reporter (IDT, diluted in water to a final concentration of 4 μM), 1 μl of Murine RNase inhibitor (NEB, #M0314), 0.8 μl of rNTP solution mix (25 mM each, NEB, #N0466), 0.6 μl of NxGen T7 RNA Polymerase (Lucigen, #30223-2) and 1 μl of MgCl₂ (120 mM). 2 μl of the RT-RPA-amplified product were then added to the mix. The 20μl-LwaCas13a reactions were transferred in 5μl-replicates (4 wells each sample) to a 384-well, round, black-well, clear-bottom plate (Corning, #3544). The plate was briefly spun down at 500 g for 15 sec to remove potential bubbles and placed into a preheated GloMax® Explorer plate reader (Promega) at 37 °C. Fluorescence was measured every 5 min for 3 h. Data analysis, if not otherwise stated, was performed at the 30-min time-point. The reporter sequence is provided in Supplementary Table 6.

Competition binding assays were performed by using an enzyme fragment complementation (EFC) method described in the HitHunter (Freemont, CA) EFC Estrogen Chemiluminescence Assay kit according to the manufacturer’s instructions. Briefly, competing ligands at final concentrations ranging from 25 pM to 2 µM were incubated with 5 nM recombinant ERα (Invitrogen) and 17β-estradiol-conjugated enzyme donor for 1.5 h. The enzyme acceptor was then added followed by the chemiluminescence substrate and incubated for 1 h. Relative luminescence was determined by using a GloMax Explorer plate reader (Promega). Sigmoidal standard curves were created by Excel^{23 (link)}.