Ez link sulfo nhs lc lc biotin kit

To determine binding affinity of ACE2, SARS-CoV-2 S trimer and the variants proteins were first subjected to gel filtration chromatography using a Superose 6 increase 10/300 GL column (GE Healthcare) pre-equilibrated with PBS and then biotinylated using the EZ-Link™ Sulfo-NHS-LC-LC-Biotin kit (Thermo Fisher) and purified using Zeba™ spin desalting column (Thermo Fisher). Biotinylated SARS-CoV-2 S trimer and variants proteins were loaded onto streptavidin (SA) biosensors (Pall FortéBio). The biosensors were dipped into wells containing varying concentrations of ACE2 protein. For WT S, Kappa S and Beta S, ACE2 concentration range used was 200–0.823 nM, while for G614S variant, ACE2 concentration range was 1800 to 7.41 nM, since at ACE2 concentration of 7.41 nM, the signal value was already close to 0. The interactions were monitored over a 500-s association period. Finally, the sensors were switched to dissociation buffer (10 mM PBS, 0.02% Tween 20 and 0.1% bovine serum albumin) for a 500-s dissociation phase. The data were corrected by subtracting the reference sample and then fitted to a 1:1 binding model for the determination of affinity constants using the software Octet Data Analysis 11.0.

The recombinant chimera CD14-Fc protein was biotinylated using the EZ-Link Sulfo NHS-LC-LC-Biotin kit (Thermo Fisher) and a fivefold molar excess of biotin. In brief, CD14-Fc was dissolved in PBS at 1 mg/ml and biotinylated with a fivefold molecular excess of biotin for 30 min at room temperature (RT). The biotinylation reaction was stopped with 3 mM Tris–HCl (pH 7.0).
The PVDF (polyvinylidene difluoride) membrane was activated for 5 min with MeOH, washed for 5 min in water and allowed to dry for 5 min. 20 ng biotinylated CD14 and roughly 20 µg type 1 pili extract from CFT073 ON (KT179) and CFT073 ON ΔfimH (KT193) mutants were loaded onto the membrane. Protein spots were allowed to dry for 10 min and then the membrane was blocked in 3% bovine serum albumin (BSA) in PBS for 30 min at 37°C. 40 ng biotinylated CD14 in 3% BSA in PBS and 0.05% Tween was blotted on the membrane and incubated for 1 hr at 37°C. Then the membrane was washed 3× in PBS with Tween for 5 min each. Streptavidin–HRP antibody was pre-diluted 1:100 in 3% BSA in PBS with Tween and diluted once more 1:5000 in PBS with Tween. The membrane was incubated with streptavidin–HRP for 1 hr at RT. After washing again 3× as before, chemiluminescence was detected using clarity ECL substrates (BioRad).

Prior to BLI assay, purified recombinant S trimer proteins of the Delta and G614 SARS-CoV-2 variants were first subjected to gel filtration chromatography using a Superose 6 increase 10/300 GL column (GE Healthcare) pre-equilibrated with 20 mM Hepes pH 7.5, 200 mM NaCl, and then biotinylated using the EZ-Link™ Sulfo-NHS-LC-LC-Biotin kit (Thermo Fisher), and Zeba™ spin desalting columns (Thermo Fisher) were then used to remove excess biotin. Binding affinities of S trimers to ACE2 were tested on an Octet Red96 instrument (Pall FortéBio, USA) according to manufacturer’s protocol. Briefly, biotinylated S trimers were loaded onto streptavidin (SA) biosensors (Pall FortéBio) until saturation. The S-immobilized biosensors were dipped into wells containing different concentrations of ACE2 monomer protein and then incubated for 500 s. Next, the biosensors were dipped into dissociation buffer (0.01 M PBS with 0.02% Tween 20 and 0.1% bovine serum albumin) and incubated for 500 s. The data were corrected by subtracting reference sample and then fitted to a 1:1 binding model for determination of affinity constants using the software Octet Data Analysis 11.0.