Pierce ms compatible magnetic ip kit

Drosophila S2 cells were transfected with 3X-FLAG-Nup358 DNA for 24 hours, followed by transfection with in vitro transcribed RNA encoding GFP, CrPV-1A or CrPV-1A(R146A) for 16 hours. Cells were washed with PBS, lysed in the Pierce-MS Compatible Magnetic IP Kit lysis buffer, and freeze thawed three times to extract the total protein. After centrifugation, the supernatant was collected, and protein concentrations were determined by Bradford assay (Biorad). Co-immunoprecipitation was performed using Pierce-MS Compatible Magnetic IP Kit (Thermofisher) according to the manufacturer’s protocol with minor modifications. Briefly 25 μL magnetic beads were incubated with 5 μg mouse monoclonal FLAG antibody (Sigma) at 4°C overnight. The beads were washed 2X times with an IP Lysis buffer and incubated with 1 mg total protein for 2 hours at 4°C overnight. Unbound proteins were washed off using wash buffers and the beads complexed with immunoprecipitated proteins were resuspended in the Pierce-MS Compatible Magnetic IP Kit elution buffer and processed for western blotting analysis.

For mass spectrometry analysis of SARS-CoV-2-S specific precipitates, individual sera containing FcγRIII/CD16-reactive soluble immune complexes were subjected to immune precipitation (IP) using Pierce MS-compatible magnetic IP kit (ThermoFisher Scientific, Darmstadt, Germany) according to manufacturer’s protocol. Briefly 250 µl serum was incubated overnight at 4 °C with 5 µg of biotinylated anti-RBD-specific TRES-1-224.2.19 mouse monoclonal antibody (1:20) or TRES-II-480 (isotype control; (1:80) (kind gift of H.M. Jäck, Erlangen) before addition of streptavidin magnetic beads. Beads were subsequently collected via centrifugation and elution buffer was added to detach putative precipitated antigen. The elution was dried in a speed vacuum concentrator and shortly run into 10% polyacrylamide gels. After over-night fixation (40% ethanol, 10% acetic acid, 50% water) and washing (3x), complete lanes were excised. Antibody biotinylation was performed using a Pierce antibody biotinylation Kit for IP (ThermoFisher Scientific, Darmstadt, Germany) according to manufacturer’s protocol.

Immunoprecipitation was performed using 200 μg of protein and using Pierce MS-Compatible Magnetic IP Kit, protein A/G (90409, Thermo Fisher Scientific) following manufacturer instructions in SDS-free conditions. Protein extracts were pulled down using 10 μg BMI1 antibody (39993, Actif Motif) or 10 μg RYBP antibody (AB3637, Millipore). Protein eluates diluted in 40 μl of 100 mM AMBIC buffer (Sigma) were analysed with MS. Extensively validated antibodies were used (45–48 (link)). For IP experiments, unbound samples were analysed to confirm sensitivity of the beads. For MS experiments proteins unspecifically binding to beads were subtracted by comparing BMI1-IP samples with mock-IP samples without antibody for each condition.

TAMRA labeled proteins were immunoprecipitated using Pierce MS-Compatible Magnetic IP Kit (Thermo Fisher Scientific, Waltham, MA) according to manufacturer’s instructions. Briefly, the labeled protein solution was precleared against washed protein A/G magnetic beads (20 μL/200 μg of protein) at 4°C for 1 h. After bead separation, the supernatant was collected and incubated with an anti-TAMRA antibody (Thermo Fisher Scientific, Waltham, MA) at 10 μg/200 μg of protein at 4°C overnight. The samples were then added to pre-washed protein A/G magnetic beads (25 μL) for 1 hour. The beads were collected and washed three times with IP-MS Wash Buffer. After washing, the beads were mixed with 100 μl elution buffer for 10 min. The supernatant was collected and dried for MS analysis.

Co-IP assays were performed with the Pierce MS-Compatible Magnetic IP Kit (Thermo Fisher Scientific) according to the manufacturer’s protocol. Briefly, after 48 h of transfection, cell lysates were incubated with an anti-c-Myc monoclonal antibody (9E10, 0.5 mg/mL, Thermo Fisher Scientific) at 4 °C overnight. After repeated washing, samples were eluted and analysed by immunoblotting with anti-Myc (9B11), anti-HA (C29F4), and anti-GAPDH (Ab9485, Abcam) antibodies. Twenty micrograms of cell lysates were analysed in the input.

Cells were lysed in IP lysis buffer and centrifuged to obtain cell lysate in the supernatant. The protein concentration was then determined using a BCA assay kit (ThermoFisher). For each reaction, 2 mg protein lysate was mixed with a specific antibody or normal IgG control in a ratio of 1000∶1 and incubated overnight at 4 °C to allow the immunocomplex to form. Pierce MS‐compatible magnetic IP kit (ThermoFisher) was used to perform the pull‐down. The beads were mixed homogenously with gentle vortexing and 25 µL was used in each reaction after two washes in IP lysis buffer. The antigen‐antibody complex was incubated with the pre‐washed beads at 4 °C for 3 h. A magnetic stand was used to collect the beads. The beads were washed three times in wash buffer A and another two times in wash buffer B, and the proteins bound to the beads were then eluted using 2x Laemmli loading buffer supplemented with 10% β‐ME at 95 °C for 10 min. Proteins were subjected to SDS‐PAGE and transferred to PVDF membrane for the immunoblotting analysis. The detailed Western blotting procedures are described in the supporting information. For the mass spectrometry analysis, proteins were stacked at the border between the stacking gel and the separating gel and the gel slices were excised and submitted to HKU proteomics and metabolomics core.