Equal numbers of 293T cells were plated and transfected the next day with HA-HUNK, GFP-Atg14L, Flag-Beclin-1, Flag-UVRAG, Flag-Rubicon, and Flag-Vps34. Equal protein was used to immunoprecipitate HUNK from cell lysates using HA or Flag antibody coupled to resin (protein A (Biorad, Des Plaines, IL, USA), protein G sepharose (Biorad), Flag resin (Sigma)), in lysis buffer and protease/phosphatase inhibitors. Lysates rotated overnight at 4°C and the next day beads were washed 3–4 times in lysis buffer prior to being prepared for immunoblotting analysis.
Protein g sepharose
Manufactured by Bio-Rad
Sourced in United States
Protein G Sepharose is an affinity chromatography resin composed of Protein G immobilized on cross-linked agarose beads. Protein G is a bacterial protein that binds to the Fc region of immunoglobulins, making it useful for the purification of antibodies from complex mixtures.
Automatically generated - may contain errors
4 protocols using protein g sepharose
1
HUNK Protein Interactions in Autophagy
2
Generation of Anti-B7-H3 Monoclonal Antibody
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8H9 hybridoma, secreting anti-B7-H3 mAb, was purchased from ATCC (USA) and cultivated according to the recommendation of the supplier. mAb was purified from the culture supernatants by affinity chromatography column with protein G-Sepharose (BioRad) and dialyzed in PBS. Concentration was determined by Bradford assay. mAb was conjugated with FITC (anti-B7-H3-FITC mAb) or Alexa Fluor 647 using a Fast kit of conjugation (Abcam).
3
Extraction and Purification of Antibodies from Agroinfiltrated Nicotiana benthamiana Leaves
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Agroinfiltrated Nicotiana benthamiana leaves (30 g for sec‐Ab, 10 g for ER‐Ab and vacs‐Abs) were grounded with mortar and pestle in liquid nitrogen until a fine powder. Then, the tissue powder was extracted with extraction buffer (1.5 m NaCl, 45 mm Tris, 1 mm EDTA, 40 mm ascorbic acid, pH 7.5) for 15 min at 4 °C with agitation, using a ratio of 1 mL buffer per 1 g fresh leaf tissue. The leaf extracts were centrifuged three times at 10 000 g for 10 min at 4 °C. The supernatant was incubated with 20 μL of Protein G Sepharose (#17‐0618‐01GE Healthcare Life Science Argentina S.A.) for 1 h 30 min at 4 °C in gentle agitation. Subsequently, antibodies bound to Protein G Sepharose were retained in Micro Bio‐Spin columns (#732‐6204; Bio‐Rad, Hercules, CA), the column was washed three times with 800 μL of extraction buffer without ascorbic acid and finally elution was performed by addition of 50 μL of SDS‐PAGE sample buffer followed of heating at 95 °C for 5 min. Extracted antibodies were used in N‐glycan analysis as described below.
4
IgG Purification and Antibody Neutralization
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Plasma from the patients was inactivated at 56 °C for 45 min. 0·5 mL of plasma was mixed with 14 mL of PBS and 250 μL of protein G sepharose 4 Fast Flow (GE Healthcare Cat#17–0618) and incubated with rotation overnight at 4 °C. Samples were spun at 1250 rpm for 5 min, supernatants were removed and protein G sepharose with bound IgG was applied onto poly-prep chromatography column (Biorad Cat#7311550). The column was washed three times with PBS. IgG was eluted from the column with 2 mL of 0·1 M glycine pH 3 (AlfaAesar, Cat#J62527). Solution was neutralized with Tris pH 8·0. Buffer was exchanged to PBS on Amicon ultra-4 centrifugal unit ultracel 30 (SigmaAldrich Cat# Z740192). IgG concentration was measured on Nanodrop Denovix DS11FX. Serial dilutions of IgG from each patient were incubated with appropriate dilution of Env-pseudoviruses on 96-well plates for 60 min. After 60 min TZM-bl cells in cDMEM with 10 μg/mL of DEAE dextran were seeded on the plates with IgG and Env-pseudoviruses. 48 h later we performed luciferase assay and calculated antibody neutralization as IC50 (μg/mL).
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