Anti flag m2 agarose column

For large-scale purification both RSV full-length and truncated and HMPV full-length constructs utilized the same method. The Sf9 cell pellets were resuspended in lysis buffer (50 mM Tris pH 8.0, 200 mM NaCl, 1 mM TCEP, 10% glycerol) and were lysed by sonication. The lysate was then centrifuged, and protein complex captured with a prepacked Anti-FLAG M2 agarose column (Sigma) that had been equilibrated in lysis buffer. The protein complex was eluted with 100 µg/mL 3xFLAG peptide (Sigma) and subsequently loaded onto a Heparin HP column (Cytiva). The protein was gradient eluted (200 mM NaCl to 1 M NaCl) from the Heparin column. Additional purification utilized size-exclusion chromatography (Superose 6, Cytiva) using a buffer of 25 mM HEPES pH 7.4, 300 mM NaCl, 6 mM MgSO₄, 0.5 mM TCEP. Final protein preparations were concentrated to 2 mg/mL for RSV and HMPV full-length complexes and 1.2 mg/mL for the RSV truncated complexes. Protein preparations were flash-frozen and stored at −70 ˚C until use.

HEK293T cells (ATCC–CRL-11268) were
transfected using Lipofectamine 3000 (Thermo Fisher) with the plasmid
encoding NPC1-domain C-Flag. 36 h post transfection, cells were washed,
lysed, and collected (Cell Lytic M-C2978, Sigma-Aldrich).
Proteins
from the cell lysate were purified by affinity chromatography using
an anti-Flag-M2 agarose column according to the manufacturer’s
instructions (Sigma-Aldrich).
Detection of NPC1-domain C-Flag
protein was performed by Western
blot using an anti-Flag M2-peroxydase (1:1000) monoclonal antibody
(Sigma-Aldrich).

Elp3 protein was purified from the Elp3-FLAG strain using an anti-FLAG-M2 agarose column (Sigma–Aldrich) as described above. The in vitro KAT assay was carried out as previously described [59 (link)]. In a total volume of 25 μl, highly purified HeLa H3/H4 core histones were incubated with 2.5 μg acetyl-CoA (Sigma–Aldrich) and 10 μg purified Elp3 in HAT reaction buffer (50 mM Tris-HCl pH 8.0, 100 mM NaCl, 5 mM MgCl₂, 1 mM dithiothreitol). After 1 hour of incubation at 30°C, the mixture was treated with 2 × SDS loading dye and heated at 95°C for 10 minutes. The acetylation levels in these samples were determined using western blotting.