Expression and purification of SARS-CoV-2 6P stabilized S trimers (Hsieh et al., 2020 (link)) and constructs encoding the sarbecovirus RBDs were conducted as previously described (Cohen et al., 2020 ). Briefly, constructs were purified from supernatants of transiently transfected Expi293F cells (Gibco) by Ni2+-NTA affinity and size exclusion chromatography (SEC). Peak fractions were identified by SDS-PAGE, pooled, and stored at 4°C. IgGs were expressed, purified, and stored as described (Barnes et al., 2020b (link)). Fabs were generated by papain digestion using crystallized papain (Sigma-Aldrich) in 50 mM sodium phosphate, 2 mM EDTA, 10 mM L-cysteine, pH 7.4 for 30–60 min at 37°C at a 1:100 enzyme:IgG ratio. To remove undigested IgGs and Fc fragments, digested products were applied to a 1-mL HiTrap MabSelect SuRe column (GE Healthcare Life Sciences) and the flow-through containing cleaved Fabs was collected. Fabs were further purified by SEC using a Superdex 200 Increase 10/300 column (GE Healthcare Life Sciences) in TBS before concentrating and storage at 4°C.
Crystallized papain
Manufactured by Merck Group
Sourced in Germany
Crystallized papain is a lab equipment product manufactured by Merck Group. It is a naturally occurring enzyme derived from papaya latex, purified and crystallized for use in laboratory settings. The core function of crystallized papain is to serve as a proteolytic enzyme for various analytical and experimental applications.
Automatically generated - may contain errors
Lab products found in correlation
Superdex 200 increase 10 300 column, by GE Healthcare (2 mentions)
Hitrap mabselect sure column, by GE Healthcare (2 mentions)
Expi293f cells, by Thermo Fisher Scientific (2 mentions)
Microtube, by Sarstedt (1 mentions)
Papain extraction reagent, by Merck Group (1 mentions)
Cystein hcl, by Merck Group (1 mentions)
Na2edta, by Carl Roth (1 mentions)
Sodium acetate, by Merck Group (1 mentions)
Blyscan assay, by Biocolor (1 mentions)
Bulletkit, by Lonza (1 mentions)
Papain, by Biocolor (1 mentions)
3 protocols using crystallized papain
1
Expression and Purification of SARS-CoV-2 Stabilized S Trimers
2
Chondrogenic Differentiation of hES-MP Cells
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Chondrogenic differentiation was performed in pellet cultures. First, 2.5 × 105 hES-MP cells were seeded in hMSC Chondrogenic Differentiation Media (BulletKit®, Lonza) in 1.5 mL microtubes (Sarstedt, Nümbrech, Germany). The tubes were then centrifuged at 150× g for 5 min to generate a cell pellet. The tube lids were then punctured with a 2 mm sterile needle (Misawa, Tokyo, Japan) to facilitate gas exchange. The medium was fully changed every 2 to 3 days. Chondrogenic pellets were harvested after 0, 7, 14, 28, and 35 days for both gene expression analysis and the evaluation of glycosaminoglycan (GAG) concentration. For GAG analysis, the pellets were washed with PBS three times and then digested for 3 h at 65 °C in a papain extraction reagent containing 0.1 M sodium acetate (Sigma-Aldrich), 0.01 M Na2EDTA (Carl Roth GmbH + Co. KG, Karlsruhe, Germany), 0.005 M cystein HCl (Sigma), and 8 µL of crystallized papain (Sigma-Aldrich). After papain digestion, the GAG concentration was evaluated with a Blyscan assay (Biocolor, Carrickfergus, UK) following the manufacturer’s instructions. The pellets were stained with hematoxylin and eosin staining and Masson’s trichrome staining after 28 days of differentiation.
3
Expression and Purification of SARS-CoV-2 S Trimers
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+ Expand
Expression and purification of SARS-CoV-2 6P stabilized S trimers (Hsieh et al., 2020 (link)) and constructs encoding the sarbecovirus RBDs were conducted as previously described (). Briefly, constructs were purified from supernatants of transiently transfected Expi293F cells (GIBCO) by Ni2+-NTA affinity and size exclusion chromatography (SEC). Peak fractions were identified by SDS-PAGE, pooled, and stored at 4°C. IgGs were expressed, purified, and stored as described (Barnes et al., 2020b (link)). Fabs were generated by papain digestion using crystallized papain (Sigma-Aldrich) in 50 mM sodium phosphate, 2 mM EDTA, 10 mM L-cysteine, pH 7.4 for 30-60 min at 37°C at a 1:100 enzyme:IgG ratio. To remove undigested IgGs and Fc fragments, digested products were applied to a 1-mL HiTrap MabSelect SuRe column (GE Healthcare Life Sciences) and the flow-through containing cleaved Fabs was collected. Fabs were further purified by SEC using a Superdex 200 Increase 10/300 column (GE Healthcare Life Sciences) in TBS before concentrating and storage at 4°C.
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