EMSAs were carried out using IR700-labeled oligonucleotides ordered from Integrated DNA Technologies (IDT) and recombinant EBNA1 protein from Abcam (ab138345). Labeled oligonucleotides were dimerized by dilution to 20 pmol/μl in 1× Tris-EDTA (TE), heating to 100°C for 5 min, and cooling slowly to room temperature. Indicated amounts of the EBNA1 protein and 1 μl of a 1:100 dilution of dimerized oligonucleotides was used for the EMSAs. Five percent polyacrylamide gels were used for electrophoresis, and gels were visualized using a Li-Cor Odyssey imager. More details, including sequences of the oligonucleotides used (Table S3 ), are provided in the supplemental material.
Ab138345
Manufactured by Abcam
Sourced in United Kingdom
Ab138345 is a polyclonal antibody that recognizes the protein Angiotensin-converting enzyme 2 (ACE2). ACE2 is an enzyme that plays a key role in the regulation of the renin-angiotensin system and is involved in various physiological processes.
Automatically generated - may contain errors
3 protocols using ab138345
1
EBNA1 Protein-DNA Binding Assay
2
EBNA1 Dimerization Assay with Zinc
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Full-length EBNA1 (residues 1 to 641) with an N-terminal His tag (ab138345; Abcam) was used in this assay. Each 0.875 µg of EBNA1 was incubated without or with 50 µM Zn2+ at room temperature in the presence of 8 ng oriP DNA and 100 µM probe (buffer/L 2 P 4 /ZRL 5 P 4 ) for 1 h at 37 °C to allow self-association to occur. After incubation, sodium dodecyl sulfate (SDS) loading buffer was added to each system, which was then separated using denaturing SDS/polyacrylamide gel electrophoresis, transferred onto a nitrocellulose membrane, and blotted with an antibody against the His tag (GeneTex); the obtained protein bands provided information of dimerization/oligomerization inhibition.
3
Antigen Immobilization on Magnetic Beads
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The viral peptides and proteins were immobilised on the surface of colour‐coded magnetic beads (MagPlex, Luminex Corp., Austin, TX, USA) as previously described.37 Briefly, each antigen was diluted to a final concentration of 80 µg mL−1 in 100 mm 2‐(N‐morpholino) ethanesulfonic acid buffer, pH 4.5 (Sigma‐Aldrich) and coupled to one bead identity (colour code). The carboxylated surface of 1 × 106 colour‐coded magnetic beads per bead identity was activated by using 100 µL phosphate buffer complemented with 0.5 mg 1‐(3‐dimethylaminopropyl)‐3‐ethylcarbodiimide hydrochloride (ProteoChem, Inc., Hurricane, UT, USA) and 0.5 mg N‐hydroxysulfosuccinimide (Thermo Fisher Scientific). Activated beads were incubated for 2 h with the diluted antigen, followed by overnight incubation in blocking buffer (Blocking Reagent for ELISA, Roche, supplemented with 0.1% (v/v) ProClin, Sigma‐Aldrich). The bead identities were then pooled to form the antigen‐bead array. Besides the viral antigens, two bead identities were coupled with anti‐human IgG (309‐005‐082, Jackson Immunoresearch, West Grove, PA, USA) and the EBNA1 protein (ab138345, Abcam, Cambridge, UK) from the common Epstein–Barr virus and used as sample loading controls.
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