Immunogens included WT-VLPs, SOS-VLPs, bald-VLPs, recombinant JR-FL gp120 and HXB2 gp41. Some animals were immunized with VLPs mixed with polyclonal IgG or with polyclonal IgG alone (e.g. guinea pig groups G2-III and G2-V, respectively, in Fig. 2 ). This polyclonal IgG was isolated by protein A-agarose chromatography (GE Healthcare) from the immune sera of an initial round of immunizations. It was then adjusted to 10mg/ml to approximately match the IgG concentration of the parent sera. For animals receiving VLP-IgG complexes, VLP pellets were resuspended in purified serum IgG and incubated for 1h at room temperature (RT). Macaque group M3 animals received "SOS trimer VLPs" that had been protease digested to clear non-functional Env (Fig. 2 ). Total immunogen volumes were 0.3ml for guinea pigs and 0.5ml for rabbits and macaques. When more than one type of masking IgG was used (e.g. group R2-II), total immunogen volumes comprised of equal volumes of each 10mg/ml IgG stock.
Protein a agarose chromatography
Manufactured by GE Healthcare
Sourced in Sweden
Protein A-agarose chromatography is a laboratory technique used for the purification and separation of proteins. It utilizes the specific binding interaction between protein A, a bacterial surface protein, and the Fc region of immunoglobulins (antibodies). This chromatographic resin allows for the efficient capture and recovery of antibodies and other Fc-containing proteins from complex mixtures.
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Lab products found in correlation
2 protocols using protein a agarose chromatography
1
Immunogens and Masking IgG in VLP Vaccine
2
Heterodimeric scFv-Fc Protein Expression
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The isolated FcCH3A and FcCH3B variant genes were subcloned in frame into the pcDNA3.1-scFv-FcCH3A and pcDNA3.1-FcCH3B vectors to express scFv-FcCH3A (hAY4 scFv-hinge-CH2-CH3A) and FcCH3B (hinge-CH2-CH3B), respectively, in mammalian cells [8 (link), 18 (link)]. To coexpress scFv-FcCH3A/FcCH3B, the two plasmids encoding scFv-FcCH3A and FcCH3B were transiently cotransfected at the indicated molar ratios with polyethylenimine (25-kDa; Polyscience) into 30–200 mL of HEK293F cells in FreeStyle 293 media (Invitrogen) as described previously [8 (link)]. After 6 days of culture, the proteins were purified from the culture supernatants using Protein-A agarose chromatography (GE Healthcare, Uppsala, Sweden) [25 (link)]. The purified proteins were analyzed by SDS-PAGE to estimate the heterodimerization yields as described previously [8 (link), 18 (link)].
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