HEK293 cells were lysed in BC200 (25 mM Tris pH 7.5, 200 mM NaCl, 1 mM EDTA, 0.2% Triton X-100, 0.2% Glycerol). Lysates were sonicated, centrifuged, and then pre-cleared using Pierce™ protein A/G magnetic beads (ThermoFisher Scientific). Supernatants were incubated with PTEN (6H2.1) or mouse IgG crosslinked to Pierce™ protein A/G magnetic beads using dimethyl adipimidate (ThermoFisher Scientific). Beads were washed four times with BC200 and proteins were eluted with 0.1 M glycine pH 2.0.
Pierce protein a g magnetic beads
Manufactured by Thermo Fisher Scientific
Sourced in United States, United Kingdom, China
Pierce Protein A/G Magnetic Beads are magnetic particles coated with a combination of Protein A and Protein G. They are designed for the rapid and efficient purification of antibodies from a variety of sample types.
Automatically generated - may contain errors
Lab products found in correlation
Anti m6a antibody, by Synaptic Systems (6 mentions)
Pierce ip lysis buffer, by Thermo Fisher Scientific (6 mentions)
Ripa buffer, by Thermo Fisher Scientific (5 mentions)
Ribominus eukaryote kit v2, by Thermo Fisher Scientific (4 mentions)
Protease inhibitor cocktail, by Roche (4 mentions)
Dynabeads mrna purification kit, by Thermo Fisher Scientific (4 mentions)
Trizol reagent, by Thermo Fisher Scientific (4 mentions)
Ip lysis buffer, by Beyotime (4 mentions)
Qiaquick pcr purification kit, by Qiagen (4 mentions)
M6a antibody, by Synaptic Systems (3 mentions)
Ripa lysis buffer, by Beyotime (3 mentions)
Agencourt ampure xp, by Beckman Coulter (3 mentions)
Sybr greener qpcr supermix universal, by Thermo Fisher Scientific (3 mentions)
Laemmli sample buffer, by Bio-Rad (3 mentions)
Igg isotype control, by Cell Signaling Technology (3 mentions)
Anti flag m2 antibody, by Merck Group (3 mentions)
Anti m6a primary antibody, by Synaptic Systems (3 mentions)
Rna fragmentation kit, by Thermo Fisher Scientific (3 mentions)
Oligo clean concentrator kit, by Zymo Research (3 mentions)
Rneasy mini kit, by Qiagen (3 mentions)
224 protocols using pierce protein a g magnetic beads
1
PTEN Immunoprecipitation from HEK293 Cells
2
CD2v Variant Immunoprecipitation and Glycosylation
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About 10 × 106 COS-1 cells were transfected with 1 µg/1 × 106 cells of the specific vector using Metafectene transfection reagent (Biontex). At 6 hpt, cells were infected with VV-T7 (MOI = 0.5 PFU/mL). At 16 hpi, cells were collected and lysed with 1 mL of Pierce IP Lysis (ThermoFisher Scientific) and protease and phosphatase inhibitors for 2 h on ice. Lysates were centrifuged and precleared with Pierce Protein A/G Magnetic Beads (ThermoFisher Scientific). CD2v variants were immunoprecipitated using either Pierce Anti-c-Myc Magnetic Beads or anti-CD2v bonded to magnetic beads Pierce Protein A/G Magnetic Beads (ThermoFisher Scientific), as indicated, at 4 °C with rotation overnight. Different immunoprecipitates were eluted and non-treated or treated with either endoglycosidases PNGase-F or Endo-H (New England BioLabs). Briefly, IP elution was denaturalized using denaturalized buffer at 100 °C for 10 min and then chilled on ice, centrifuged at 11,000 rpm, and incubated with PNGase-F or Endo-H (1 h at 37 °C). Finally, samples were analyzed by Western blot.
3
Magnetic Bead-based Depletion of Wnt and IFNγ
4
Immunoprecipitation Protocol for TM4SF19 Protein
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For immunoprecipitation analysis, RAW264.7 macrophages overexpressing Myc-fused TM4SF19 were harvested by lysis buffer (50 mM Tris-HCl (pH 7.6), 150 mM NaCl, 2 mM EDTA, and protease inhibitor). After 10 min of incubation on ice, lysates were centrifuged at 13,000 rpm, at 4 °C for 15 min and the pellet was discarded. 50 μL of Pierce™ Protein A/G Magnetic Beads (Invitrogen, 88802) were incubated with 5 μL of Myc and IgG antibodies for 2 h rotating at 4 °C. Beads were washed once in lysis buffer before incubation with 300 μg of lysate for 4 h, rotating at 4 °C. Beads were then washed five times in a buffer containing Tris-buffered saline (TBS) containing 0.05% Tween-20 Detergent. Bound proteins were eluted by 0.1 M glycine (pH 2.0).
5
Depletion of Wnt proteins from conditioned media
6
Protein Interaction Detection Assays
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IP and WB assays were performed as previously described [26 (link)]. In brief, to detect protein interactions, cells were lysed in IP buffer. These supernatants were immunoprecipitated with the indicated antibodies, slowly shaken on a rotating shaker at 4 °C overnight, and then incubated with Pierce™ Protein A/G Magnetic beads (Invitrogen) at room temperature for 1 h. Then, the binding proteins were eluted and boiled in 1× SDS loading buffer to prepare the samples for immunoblot analysis.
7
Immunoprecipitation and Immunoblotting of Cellular Proteins
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Cell lysate (1 mg) was incubated for 1 h with IgG and 10 μg of protein A/G beads [24 (link)]. The lysate was then incubated overnight at 4 °C with 10 μg of anti-ARG2 (CST.) and anti-ARL1 (SCB) antibodies or nonspecific IgGs. Following this, the lysate was incubated once more for 1 h at RT with 20 μg PierceTM protein A/G magnetic beads (Thermo Fisher Scientific Inc.). The samples containing the beads were placed in a magnetic separation rack and then the supernatants carefully removed. Pellets were washed with 1 mL of the lysate buffer (10 Mm Tris-HCl, pH7.4, 150 mM NaCl, 5 Mm EDTA, 0.1% Triton X-100). The immunoprecipitated proteins were extracted by 10 min boiling with 2x SDS-PAGE reducing sample buffer 40 μL and detected by immunoblot with anti-HIF1A or anti-Slug antibodies
8
Antibody-Coated Magnetic Bead Capture
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Coating an anti-ApoB antibody (Lot; 2007015, Cat No; NB200527, Novus Biologicals, Littleton, CO, USA) with magnetic beads was performed by mixing 10 μL of magnetic beads (pierceTM ProteinA/G magnetic beads; Thermo Scientific) with 10 μL the anti-ApoB antibody, which was then gently agitated on a shaker at room temperature for 1 h. Anti-ApoB antibody-coated magnetic beads were washed twice with PBS using a magnet and resuspended in EV samples (4 μg in 50 μL), which were then gently agitated on a shaker at room temperature for 1 h. Next, the beads were collected using a magnet and the supernatant (non-captured fraction) was harvested. The magnet beads were washed twice with PBS and resuspended in PBS (captured fraction). The captured fraction and non-captured fraction were used for downstream assays.
9
Co-immunoprecipitation and Ubiquitination Assay
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Cells were lysed on ice with IP lysis buffer supplemented with protease and phosphatase inhibitors. The lysates were immunoprecipitated with the indicated antibodies (3 μg) overnight at 4 °C. PierceTM Protein A/G Magnetic Beads (Thermo Scientific) were used to capture the immune complexes, which were washed with IP wash buffer. The eluates were then separated by SDS–PAGE and stained with a Fast Silver Stain Kit (Beyotime, P0017S). MS was conducted by Wininnovate Biotechnology. The proteins of interest in the co-IP products were detected by Western blotting. The ubiquitin assay was conducted under denaturing conditions as previously described48 (link). The antibodies used are listed in Supplementary Table 5 .
10
Immunoprecipitation of Proteins from HUVECs
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HUVECs were cultured in a 10 cm culture dish. The cells were washed three times with PBS, and cell lysis buffer for immunoprecipitation (IP) (Beyotime, Shanghai, China) was added for 30 min. Subsequently, the mixture was centrifuged at 12,000 rpm for 10 min to obtain the supernatant. PierceTM Protein A/G Magnetic Beads (Thermo Scientific, USA) were utilized following the provided instructions. Briefly, the beads were mixed for 1 min, and each group took 50 μL of beads. The beads were washed three times with wash buffer and then incubated with the corresponding antibody at room temperature for 30 min. After three additional washes, the beads were incubated with the protein solution overnight at 4°C. On the second day, the beads were washed 3–5 times, and 1× loading buffer was added at 100°C for 10 min. Subsequently, the beads were discarded, and the supernatant was collected for Western blot analysis.
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