HEK293T cells were seeded in 10 cm plates at 1.3×106 cells/ml in 10 mL DMEM with 10% FBS and 1% penicillin streptomycin the day before transfection. 3μg of indicated ISG15 or mutant were transfected, using X-tremeGENE HP DNA transfection reagent (Roche), into one 10 cm plate and grown for 48 hours. Cell culture supernatants were collected and cleared of any cell debris by centrifugation at 1000xg. These cell culture supernatants were further cleared of IgG from the serum by two one-hour bindings with Protein A Sepharose (Invitrogen) at room temperature. 20uL of Sera-Mag SpeedBead Protein A/G magnetic beads (GE Life Sciences), 10 uL of ISG15 antibody (Invitrogen 7H29L24) and 20 mL of 0.1% NP40 in PBS pH 7.0 were added to the cell culture supernatants and rocked for 2 hours at room temperature. Beads were isolated with a neodymium magnet and washed 3X with 0.1% NP40 in PBS pH 7.0. ISG15 was eluted from the beads by boiling in 80μl of 1X loading dye and 40μl was run on a NuPAGE 4%–12% Bis-Tris gel and transferred to nitrocellulose for western blotting. ISG15 was detected with the M2 Flag antibody (Sigma).
Sera mag speedbead protein a g magnetic beads
Manufactured by GE Healthcare
Sera-Mag SpeedBead Protein A/G magnetic beads are a laboratory product designed for the capture and purification of antibodies. They consist of superparamagnetic beads coated with recombinant Protein A and Protein G. These beads facilitate the efficient and rapid separation of antibodies from complex biological samples using a magnetic field.
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2 protocols using sera mag speedbead protein a g magnetic beads
1
ISG15 Secretion Quantification Assay
2
ISG15 Immunoprecipitation and Detection
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HEK293T cells were seeded in 10 cm plates at 1.3x106 cells/ml in 10 mL DMEM with 10% FBS and 1% penicillin streptomycin the day before transfection. 3μg of indicated ISG15 or mutant were transfected, using X-tremeGENE HP DNA transfection reagent (Roche), into one 10 cm plate and grown for 48 hours. Cell culture supernatants were collected and cleared of any cell debris by centrifugation at 1000xg. These cell culture supernatants were further cleared of IgG from the serum by two one-hour bindings with Protein A Sepharose (Invitrogen) at room temperature. 20uL of Sera-Mag SpeedBead Protein A/G magnetic beads (GE Life Sciences), 10 uL of ISG15 antibody (Invitrogen 7H29L24) and 20 mL of 0.1% NP40 in PBS pH 7.0 were added to the cell culture supernatants and rocked for 2 hours at room temperature. Beads were isolated with a neodymium magnet and washed 3X with 0.1% NP40 in PBS pH 7.0. ISG15 was eluted from the beads by boiling in 80μl of 1X loading dye and 40μl was run on a NuPAGE 4%–12% Bis-Tris gel and transferred to nitrocellulose for western blotting. ISG15 was detected with the M2 Flag antibody (Sigma).
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