Pierce™ Protein A/G magnetic agarose beads (Thermo Fisher Scientific, Waltham, MA, USA) were prepared according to the manufacturer's protocol. To deplete IgG, beads were incubated with serum from human donors, previously shown to be positive for AAV9-induced complement activation. To obtain IgG/IgM-depleted serum, the IgG-depleted serum was then incubated with anti-human IgM−agarose antibody (Sigma, St. Louis, MO, USA), and then centrifuged. AAV9 (empty capsid)-induced complement activation was assessed as described above.
Pierce protein a g magnetic agarose beads
Manufactured by Thermo Fisher Scientific
Sourced in United States
Pierce™ Protein A/G Magnetic Agarose Beads are magnetic agarose beads designed for the purification of antibodies and immunoglobulins from biological samples. The beads contain a covalently immobilized Protein A/G ligand, which binds to the Fc region of immunoglobulins.
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4 protocols using pierce protein a g magnetic agarose beads
1
Depletion of IgG and IgM for AAV9 complement assessment
2
Purification of Flag-tagged Proteins
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For purification of Hsc70-WT-Flag, Hsc70-S85A-Flag, Lamp2a-Flag, APP-WT, and APP-M7, pcDNA5-Hsc70-WT-Flag, pcDNA5-Hsc70-S85A-Flag, pcDNA5-Lamp2a-Flag, pcDNA5-APP, and pcDNA5-APP-M7 plasmids were transfected into HEK293T cells or SH-SY5Y cells for 24 h. Cells were washed with PBS and lysed in 1 mL of lysis buffer (TAP) (20 mmol/L Tris-HCl (pH 7.5), 150 mmol/L NaCl, 0.5% NP-40, 1 mmol/L NaF, 1 mmol/L Na3VO4, 1 mmol/L EDTA, Protease inhibitor cocktail (Bimake, add fresh) for 30 min, incubated with anti-Flag (DYKDDDDK) beads or APP antibodies incubated with Pierce™ Protein A/G Magnetic Agarose Beads (#78610, Thermo Fisher Scientific) for 6 h on a rotating wheel at 4 °C. The beads were washed with TAP three times, 5 min each wash, and SDS-PAGE 2× loading buffer was added, followed by heating at 100 °C for 10 min.
3
Immunoprecipitation of Protein Complexes
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Transfected cells were lysed with 200 µl of RIPA buffer (Beyotime Institute of Biotechnology) containing PMSF and a protease inhibitor. The lysate (800 µl) was subsequently incubated with 2 µg of antibody against SENP1 (ProteinTech Group, Inc.; cat. no 25349-1-AP), β-catenin (Beyotime Institute of Biotechnology; cat. no. AC106), Flag (MilliporeSigma; cat. no. F2555) or IgG (Abcam; cat. no. ab6715) with gentle rotation overnight at 4°C, before being subsequently incubated with Pierce™ Protein A/G Magnetic Agarose Beads (20 µl; Thermo Fisher Scientific, Inc.; cat. no. 78609) for 2 h. The beads coupling with the immune-complexes were centrifuged for 3 min at 4°C and 200 × g to sink the agarose bead to the bottom of the tube. The supernatant was removed carefully, and the agarose beads were washed with lysis buffer before the proteins were eluted in SDS-PAGE buffer with centrifugation at 1,000 × g for 1 min at room temperature. Thereafter, eluted proteins were separated on 10% gels using SDS-PAGE. The interacting proteins were detected by western blot analysis.
4
Immunoprecipitation of H3 and Modifications
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After 48 h, the transfected F9 ECs were washed with PBS twice and lysed in ice-cold Pierce IP lysis buffer (Pierce, Rockford, USA) supplemented with proteinase inhibitor cocktail (Thermo Scientific, Waltham, USA). Cleared cell lysates (0.5 mL) were pre-incubated with Pierce™ Protein A/G Magnetic Agarose Beads (Thermo Scientific) for 1 h at 4°C to reduce non-specific binding of proteins to the beads. After brief spin, the pre-cleared cell lysates were incubated with 2 μg of indicated antibodies overnight at 4°C, and the lysates were added in Pierce™ Protein A/G Magnetic Agarose Beads and incubated for 3 h. Then the immunoprecipitates were analyzed by western blot analysis. The biotin co-immunoprecipitation was preformed using bio-peptides of H3, H3K9me2 and H3K9me3 in HEK-293T cells with overexpression of PGC7 and UHRF1, the Streptavidin Dynabeads™ M-280 were used to pull down the complex that bio-peptides bound to. The information bio-peptides is as follows: L-H3, biotin-ARTKQTARKSTGGKAPRKQLA; L-H3K9me2, biotin-ARTKQTARK(me2)STGGKAPRKQLA; L-H3K9me3, biotin-ARTKQTARK(me3)STGGKAPRKQLA.
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