After transient transfection GFP labeled plasmids into HEK293T cells, the cells were lysed as described before. The magnetic beads (Beyotime, Shanghai, China) were incubated with the GFP antibody at room temperature for 2 h. The magnetic beads were then incubated with cell extracts (1 mg) at 4 °C for overnight, collecting magnetic beads with a magnetic separation rack. The beads were washed in lysis buffer, mixed with 5× Loading buffer, boiled 25 min, and detected by WB.
Magnetic beads
Manufactured by Beyotime
Sourced in China
Magnetic beads are spherical particles that can be attracted to a magnetic field. They are made of a magnetic material, such as iron oxide, and are coated with a polymer or other material to provide a surface for various applications. Magnetic beads are used in a variety of laboratory applications, including cell separation, protein purification, and nucleic acid extraction.
Automatically generated - may contain errors
Lab products found in correlation
Ip lysis buffer, by Thermo Fisher Scientific (1 mentions)
Ip lysis buffer, by Cell Signaling Technology (1 mentions)
Magnetic separation rack, by Cell Signaling Technology (1 mentions)
Protein a g magnetic beads, by Beyotime (1 mentions)
Immunoprecipitation lysis buffer, by Beyotime (1 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Beyotime (1 mentions)
Protease and phosphatase inhibitor, by Beyotime (1 mentions)
Magnetic stand, by Thermo Fisher Scientific (1 mentions)
8 protocols using magnetic beads
1
GFP-Labeled Protein Immunoprecipitation
2
Immunoprecipitation and Western Blot
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According to the instructions for the magnetic beads (Beyotime, Shanghai, China), 500 μL of protein sample and 20 μL of magnetic beads suspension were prepared and then incubated at 4°C overnight. The magnetic beads were removed the next day. The antibodies were added, and the samples were incubated for 2 h before being washed again. Then, 100 μL of buffer was added to the sample, heated at 95°C for 5 min. These samples were subjected to western blot analysis.
3
Immunoprecipitation and Protein Detection
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First, the magnetic beads (Beyotime, Shanghai, China) were incubated with the indicated antibody at room temperature for 2 h, then wash and collect the beads and added into the prepared cell lysate lysed in IP Lysis Buffer (Thermo USA), incubated overnight at 4 °C. The immunoprecipitates were washed 4–5 times with IP Lysis Buffer before being collected by magnetic separation rack (Cell Signaling). Add 60 μl IP Lysis Buffer and 5× SDS-PAGE loading buffer, boil for 25 min and collect samples, immunoblotted with the indicated antibodies. Detecting the proteins with enhanced chemiluminescence (Pierce, Rockford, IL, USA).
4
Cell Lysis, Immunoprecipitation and Western Blot
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The lysis of cells was operated on ice for 10 min, following which was the centrifugation at 13,000 x g for 10 min and the collection of supernatants. Subsequently, the lysate (500 µg/ IP) was supplemented with the 2.5 µg CUL4A or PRMT5 antibody and 10 µl protein A + G magnetic beads (Beyotime, Shanghai, China) followed by gentle rotation at room temperature for 2 h. The supernatant was removed by magnetic force and the magnetic beads with the added 1X SDS sample buffer were boiled for 5 min for western blotting [23 (link)].
5
Immunoprecipitation and Western Blotting
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Cell lysates were obtained on the required day by using immunoprecipitation lysis buffer (cat#: P0013, Beyotime) containing 1 % phosphatase inhibitors and 1 % protease inhibitors. Then, the supernatant was obtained as described for western blotting. Part of the supernatant was added to SDS sample loading buffer to prepare for whole-cell lysis. The remaining supernatant was added to magnetic beads (cat#: P2018, Beyotime) for precleaning. Subsequently, the supernatant was incubated with antibodies against Flag (1:50), AKT1 (1:100), PDK1 (1:50) or HDAC3 (1:50) for 3 h at 4 ℃ and then with magnetic beads overnight at 4 ℃. The antibodies used for western blotting noted above were then used as described. The magnetic beads were washed 5 times, SDS sample loading buffer was added, and then samples were boiled for 10 min. The target protein sample was finally obtained after the magnetic beads were discarded.
6
Fungal Quantification of Larvae
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An additional 160 healthy larvae of uniform size (250–350 mg) were randomly selected. Four groups of randomly selected larvae (20 for each group) were established as described above and incubated at 35°C for 4 days (Kong et al., 2020 (link)). During incubation, five larvae from each group were randomly selected daily on days 1–4. A high-speed homogenizer (Powteq GT300, Grinder Instrument, Beijing, China) was used to thoroughly grind the larvae for 30 min (1,600 rpm) after adding 0.7 mL PBS and sterile magnetic beads (3 mm, Beyotime, Haimen, China). We used PBS to dilute the homogenate in a gradient and inoculated 5 μL of samples with different concentrations on Sabouraud dextrose agar (SDA) at 37°C for 48 h.
7
Protein Extraction and Detection
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The treated cells were harvested 24 h posttransfection. Cells were lysed in wells with cell lysis buffer containing PMSF (Beyotime Biotechnology). Protein lysates were centrifuged for 15 min at 15,000 × g at 4 °C and then incubated with magnetic beads (Beyotime Biotechnology) at 4 °C for 8 h with rotation. The beads were washed and then subjected to SDS/PAGE and western blotting analysis.
8
Protein Extraction and Immunoprecipitation
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Proteins were extracted from 100 mg of tissue using 1 ml of ice-cold RIPA lysis buffer containing fresh protease and phosphatase inhibitors (Beyotime Institute of Biotechnology; cat. no. P0013D) and concentrations measured. After treatment of samples, 500 μg of proteins diluted in 500 μl of RIPA lysis buffer were incubated with primary antibody (Integrin β1: 2 μg per IP reaction; KCNH2: 2 μg per IP reaction) and goat IgG isotype control (2 μg per IP reaction) separately, and then with rotation overnight at 4°C. Magnetic beads (Beyotime Institute of Biotechnology; cat. no. P2105) were added and the mixture was shaken for another 4 h. After incubation, the mixtures were placed on a magnetic stand (Invitrogen; Thermo Fisher Scientific, Inc.) for 10 sec at 4°C to separate the beads. The bead complexes were washed with RIPA lysis buffer and then the immunoprecipitants were mixed with SDS loading buffer, boiled for 10 min and subjected to western blotting as aforementioned (see Table SII for details of antibodies).
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