Glycobuffer 2

AAV9 capsids were treated with PNGase F in GlycoBuffer 2 or enzyme buffer GlycoBuffer 2 (New England Biolabs) only as a control at 37 °C for 1 h. The samples were then denatured and reduced using TCEP at a final concentration of 20 mM in 50% (v/v) dimethyl sulfoxide (DMSO) at 70 °C for 10 min. The entire volume of the above prepared samples was used for cIEF analysis as described in [32 (link)], except the concentration of formamide was decreased to 40% by volume to increase sample concentration. The fluorescence detection default setting was excitation at 280 nm and emission at 458 ± 30 nm, and the exposure time used was 20 s. The samples were prepared independently across two different days (biological replicates); each biological replicate was injected either two or three times (each injection was considered a technical replicate). The data were analyzed in Compass and then exported to Chromeleon (Thermo Fisher Scientific) for better visualization. A standard IgG1 monoclonal antibody was used as a control and analyzed in the same way as the AAV9 capsids to ascertain the completion of deglycosylation.

Purified, untagged Pf12 D1D2 and Pf41 D1D2 proteins were mixed in a molar ratio of 1.2:1 and incubated for 1 h on ice. SEC in 20 mM HEPES pH 7.5, 150 mM NaCl was performed to separate the formed Pf12-Pf41 complex from excess Pf12. For the purpose of de-glycosylation Glyco buffer 2 and PNGase F (NEB) was added to the mix of Pf12 and Pf41, incubated at RT for 1h prior SEC in 20 mM HEPES pH 7.5, 150 mM NaCl.
Purified, untagged Pf12 D1D2 was mixed with Glyco buffer 2 and PNGase F (NEB) and incubated at RT for 1h prior the addition of Nb in a molar ratio of 1:1.5. The proteins were incubated for 1h on ice followed by SEC in 20 mM HEPES pH 7.5, 150 mM NaCl to separate the formed Pf12-Nb complex from excess Nb.

In total 10 µL of total reaction mixture contained 7.5 µL of sample, 1 µL of 10× Glyco Buffer2, 1 µL of 10% NP-40, and 0.5 µL of PNGase F (New England Biolabs). Reactions were incubated at 37 °C for 1 h. Digestion of glycans (100 nM) with 6.4 U of α1,2-3-mannosidase (New England Biolabs) and 4 U of α1,6-mannosidase (New England Biolabs) was performed at 25 °C for 16 h in 10 μL (total reaction volumes) with buffers supplied by the manufacturer.