Pierce crosslinking magnetic ip co ip kit

Pierce crosslinking magnetic IP/Co-IP kit (Thermo Scientific) was used to perform IP/Co-IP assay. Control IgG or specific antigen antibodies (5 μg) was bound to protein A/G magnetic beads through 15-min incubation at room temperature. Disuccinimidyl suberate (20 μM) was used for crosslinking the bound antibody. Cells were lysed in precooled IP wash buffer and then centrifugated at 12,000 × g for 5 min. The supernatant was incubated with various specific antibodies or irrelevant IgG in the presence of protein A/G magnetic beads for 1 h, which was on a rotator and at room temperature. After stringent washing, the proteins were eluted from the magnetic beads using elution buffer. The proteins were boiled in SDS loading buffer before SDS-PAGE.

Samples from different fermentation times were purified by a Pierce™ Crosslinking Magnetic IP/Co-IP Kit (Thermo Fisher Scientific, Waltham, MA, USA). Briefly, melatonin IgG-Dynabeads were prepared by crosslinking anti-melatonin rabbit IgG polyclonal antibody to Dynabeads (LifeSpan BioSciences, Seattle, WA, USA). Then, 10⁸ cells were resuspended in 1 mL of extraction buffer (25 mM Tris, 150 mM NaCl, 1 mM EDTA, and 1% NP40, pH 7.4) and lysed by glass beads by applying three shaking cycles of 1 min in Mini Beadbeater-24 (BioSpec Products, Bartlesville, OK, USA) and 1 min on ice. Lysed cells were centrifuged at 3,000 rpm and 4°C for 10 min to remove insoluble particles. Melatonin IgG-Dynabeads were added to each lysate, and the suspension was rotated for 1 h at room temperature. The melatonin IgG-Dynabeads were collected with a magnet and washed three times with 500 μL of ice-cold extraction buffer. Isolated proteins were eluted from the melatonin IgG-Dynabeads by following manufacture’s instructions and resolved by 12% SDS-PAGE. Proteins in the gel were visualized by Pierce™ Silver Stain kit (Thermo Fisher Scientific, Waltham, MA, USA). ImageJ software¹ was used to quantify the protein intensities in the SDS-PAGE analysis.

Pierce crosslinking magnetic IP/Co-IP kit (Thermo Scientific) was used. Prior to immunoprecipitation, binding of antibody (5 μg) to protein A/G magnetic beads was performed by incubating for 15 min at room temperature according to the manufacturer’s protocols. Disuccinimidyl suberate (20 μM) was used for crosslinking the bound antibody. Cells were lysed at 4 °C in ice-cold IP Lysis/Wash buffer and cell lysates were cleared by a brief centrifugation (12,000×g, 10 min). Concentrations of proteins in the supernatant were determined by Bradford assay. Samples containing equal amount of proteins were incubated with various irrelevant IgG or specific antibodies in the presence of protein A/G magnetic beads for 1 h on a rotator. After incubation, protein A/G magnetic beads were washed extensively with IP Lysis/Wash buffer and proteins were eluted by boiling in 5× SDS sample buffer before SDS-PAGE.