For co-immunoprecipitation assays, 4×106 HEK293 cells were seeded in 10-cm plates and transfected the next day with 5 µg Flag–GFP or Flag–NSP2 plasmids using Lipofectamine 2000 according to the manufacturer's instructions. After 24 h, cells were washed with ice-cold PBS and collected by scraping in lysis buffer (40 mM HEPES pH 7.5, 120 mM NaCl, 1 mM EDTA, 0.3% CHAPS), supplemented with complete EDTA-free protease inhibitor tablet (Roche, 11836170001)]. After a 30 min incubation on ice with end-to-end rotation, the lysates were separated from debris by centrifugation at 14,000 g for 15 min at 4°C. The supernatants were pre-cleared by incubation with 50 µl washed and blocked Dynabeads Protein G (Thermo Fisher Scientific, 10004D) for 1 h at 4°C. 2 mg pre-cleared lysate was incubated with 3 µg anti-Flag antibody, 60 µl Dynabeads Protein G and 1 µl RNase I (Invitrogen, AM2294) with end-to-end rotation at 4°C overnight. Beads were washed three times for 10 min with wash buffer (50 mM HEPES pH 7.5, 150 mM NaCl, 1 mM EDTA, 0.3% CHAPS and complete EDTA-free protease inhibitor tablet). Protein was eluted in 2× SDS sample buffer for subsequent analysis by western blotting.
Dynabeads protein g
Manufactured by Thermo Fisher Scientific
Sourced in United States, Norway, Germany, United Kingdom, China, Canada, Japan, Denmark, Australia, France
Dynabeads Protein G is a magnetic bead-based product used for the purification and isolation of immunoglobulins (Ig) and other proteins that bind to Protein G. It provides a reliable and efficient method for the capture and separation of target proteins from complex samples.
Automatically generated - may contain errors
Lab products found in correlation
Protease inhibitor cocktail, by Merck Group (42 mentions)
Protease inhibitor cocktail, by Roche (41 mentions)
Pierce ip lysis buffer, by Thermo Fisher Scientific (30 mentions)
Complete protease inhibitor cocktail, by Roche (28 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (22 mentions)
Dynabeads protein a, by Thermo Fisher Scientific (21 mentions)
Bioruptor, by Diagenode (20 mentions)
Qiaquick pcr purification kit, by Qiagen (20 mentions)
Anti flag antibody, by Merck Group (18 mentions)
Nupage lds sample buffer, by Thermo Fisher Scientific (18 mentions)
Protease inhibitor, by Roche (17 mentions)
Trizol reagent, by Thermo Fisher Scientific (16 mentions)
Proteinase k, by Thermo Fisher Scientific (16 mentions)
Trizol, by Thermo Fisher Scientific (15 mentions)
Anti flag, by Merck Group (13 mentions)
Protease inhibitor, by Merck Group (13 mentions)
Dnase 1, by Thermo Fisher Scientific (13 mentions)
Laemmli sample buffer, by Bio-Rad (12 mentions)
Normal rabbit igg, by Cell Signaling Technology (12 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (12 mentions)
1 538 protocols using dynabeads protein g
1
Co-immunoprecipitation of Flag-tagged Proteins
2
Affinity Purification of Protein Complexes
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Nuclear extracts were prepared as described previously [46 (link)]. Nuclear extracts were pre-cleared with 6 μg of normal mouse IgG (Santa Cruz Biotechnology) conjugated to 60 μl of Dynabeads-Protein G (Invitrogen) and subsequently incubated with 6 μg of anti-FLAG M2 Ab conjugated to 60 μl of Dynabeads-Protein G at 4°C for 2 h. The Dynabeads were washed five times with 1 ml of Wash Buffer (20 mM Tris pH 8.0, 150 mM NaCl, 25% glycerol, 1.5 mM MgCl2, 0.2 mM EDTA, 0.1% IGEPAL-CA630) and once with 1 ml of TBS buffer with 0.1% IGEPAL-CA630. The immunoprecipitants were eluted with 40 μl of Elution Buffer, mixed with 2 × Sample Buffer, boiled for 5 min, and subjected to SDS-PAGE. The proteins visualized by silver staining were excised and analyzed by LC-MS/MS at DNA-chip Development Center for Infectious Diseases (RIMD, Osaka University).
3
Quantitative ChIP Assay Protocol
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Quantitative ChIP assay was performed as previously described70 with some modification. HEK293T transfectants in six-well plates and MCF-7 cells (ATCC) in 12-well plates were fixed in 11% formaldehyde for 15 min, quenched with 0.125 M glycine for 10 min, and rinsed with cold 1 × PBS. The cells were disrupted in lysis buffer70 , incubated at 4 °C for 1 h and then sonicated. The lysates were then incubated with 100 μl Dynabeads protein G (Invitrogen, Thermo Fisher Scientific, Inc.) and 10 μl pre-immune rabbit IgG for 1 h at 4 °C, and centrifuged at 12,000 r.p.m. for 15 min at 4 °C. One hundred microliters of the pre-cleared supernatant was mixed with an antibody (Supplementary Table 3 ) (Santa Cruz Biotechnology, Inc.), 25 μl Dynabeads protein G (Invitrogen, Thermo Fisher Scientific Inc.), and lysis buffer to make a 200 μl IP mixture that were rotated overnight at 4 °C. The precipitates were sequentially washed in low-salt, high-salt, and LiCl buffers70 , and twice in TE buffer. The crosslinks were then reversed at 65 °C for 3 h. DNA fragments were isolated using QIAquick PCR purification kit (QIAGEN, Hilden, Germany), and analyzed by qPCR using TaqMan® 2 × master mix and custom TaqMan® real-time PCR assays (Applied Biosystems, Thermo Fisher Scientific, Inc.) (Supplementary Table 4 ).
4
Analyzing NS1B-ISG15 Interaction in A549 Cells
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For analysis of NS1B-ISG15 interaction, 3F-S-ISG15GG A549 cells were first treated with the indicated siRNA for 24 h, followed by 16 h of treatment with 1,000 IU ml−1 of human IFN-β and then infection with 5 p.f.u. per cell of either wt or 67 mutant virus. The cells were harvested at 18 h after infection, and were extracted with cold RIPA lysis buffer (50 mM Tris-HCl pH 7.5; 200 mM NaCl; 2 mM MgCl2; 0.5% NP-40; 0.5% sodium deoxycholate; 0.1% SDS) supplemented with protease inhibitor and 0.5 units μl−1 benzonase (Sigma). After brief sonication, the lysate was cleared by centrifugation at 16000xg for 10 min at 4 °C. The protein concentration of the extract was measured using the DC protein assay (Biorad), and an aliquot of the extract containing 700 μg of protein was used for co-immunoprecipitations. The extract was precleared by incubation with 50 μl Dynabeads Protein G (Invitrogen) for 2 h at 4 °C, and then incubated for 3 h with 50 ul Dynabeads Protein G (that had been preabsorbed with 10 μg of NS1B Ab). The beads were washed five times with RIPA buffer, and the bound proteins were eluted with SDS sample buffer, and analysed by immunoblots probed with NS1B or Flag Ab.
5
Co-immunoprecipitation for CHI3L1 detection
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Serum samples were obtained from patients in The First Affiliated Hospital of Bengbu Medical College, China. The samples were collected with the informed consent of the patients, and all related procedures were performed with the approval of the internal review and ethics boards of the indicated hospital. For the co-immunoprecipitation assay, the sera were centrifuged at 12,000 × g and 4 °C for 10 min. Then, the supernatants were diluted in EBC lysis buffer (50 mM Tris–HCl, 120 mM NaCl, and 2 mM PMSF). To remove the antibodies from the sera, the supernatants were incubated with Dynabeads® protein G (Invitrogen) with gentle rotation at 4 °C for 2 h. After centrifugation at 5,000 × g for 5 min, the supernatants were incubated with the anti-CHI3L1 IgG-conjugated Dynabeads® protein G with gentle rotation at 4 °C overnight. Subsequently, the mixture was washed twice using EBC lysis buffer and was analyzed by western blotting using the anti-CHI3L1 IgG.
6
RNA-Immunoprecipitation Protocol for Plants
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Before RNA-IP, 50 µl/IP of Dynabeads Protein G (Invitrogen) were washed in 1X PBS + 0.1% Tween, followed by incubation with 1 µg/IP FLAG antibody (Sigma) at room temperature for 90 min with rotation. For each sample, 0.5 g leaf tissue was crosslinked in formaldehyde and ground in liquid nitrogen. Proteins were extracted using 50 mM Tris–HCl pH 7.5, 150 mM NaCl, 5 mM MgCl2, 10% glycerol, 1% NP-40 (IGEPAL), 0.5 mM DTT, 1 mM PMSF, 1% Plant PIC (GoldBio protease inhibitor cocktail). Lysates were pre-cleared with Dynabeads Protein G (Invitrogen) with rotation for 20 min at room temperature. Pre-cleared lysates were then incubated with the prepared IP beads for 90 min at 4°C with rotation. Beads were washed 3X in the washing buffer (50 mM Tris–HCl pH 7.5, 150 mM NaCl, 5 mM MgCl2, 0.5 mM DTT). After the final wash, 1 mL Trizol LS (Invitrogen) per sample was added, reverse crosslinking was performed at 55°C for 5 min and RNA was extracted following the manufacturer’s protocol.
7
Immunoprecipitation of RNA-binding Proteins
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For the pulldown of DAP3, SFPQ and NONO protein, EC109 cells were lysed with prechilled lysis buffer (50 mM Tris-HCl, pH 7.5; 150 mM NaCl; 1% Nonidet P40; 0.5% sodium deoxycholate; 1× EDTA-free cOmplete protease inhibitor (Roche)). The lysates were precleared with Dynabeads™ protein G (Invitrogen) at 4 °C overnight. The precleared lysates with incubated with anti-DAP3 (abcam, ab2637), anti-SFPQ (Santa Cruz, sc-271796) and anti-NONO (Santa Cruz, sc-166702) antibodies for 4 h at 4 °C and subsequently with Dynabeads™ protein G at 4 °C overnight. The Dynabeads™ protein G (Invitrogen) with bound proteins were washed with 150 mM NaCl with 1× EDTA-free cOmplete protease inhibitor for six times and boiled with 2× protein loading buffer for 10 mins at 95 °C to elute bound proteins. Western blot analysis was performed to detect co-IP products. For the RNase A treatment prior to the immunoprecipitation, the total lysates were incubated with 0.1 μg/μl RNase A (Thermo Fisher Scientific) at 37 °C for 10 min.
8
RNA Immunoprecipitation and qRT-PCR Analysis
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AC16 cardiomyocytes grown in 10 cm dishes were rinsed twice with pre-ice-cold PBS and harvested. Cell pellets were resuspended in lysis buffer and flash-frozen with liquid nitrogen. Cell lysates were thawed on ice and subjected to 5 rounds of sonication to lyse the cell nucleus. Cell lysate was subjected to centrifugation at 12000g for 30 min at 4°C and precleared by binding to Invitrogen™ Dynabeads Protein G. 1% cell lysate was saved as input. 3 μg anti-rabbit-IgG and YTHDF3 rabbit polyclonal antibody (Proteintech, #25537-1-AP) were incubated with precleared cell lysate at 4°C overnight. 60 μL Invitrogen™ Dynabeads Protein G was washed with NET2 buffer supplied with 200 U/mL RiboLock RNase Inhibitor and 2 mM Ribonucleoside Vanadyl Complexes and then mixed with cell lysate and antibody for another 4 hrs. The beads were collected and washed with NET2 buffer for 4 times. Beads were mixed with 1 mL TRIzol and saved as the IP sample. RNA was isolated by the miRNeasy Mini Kit (QIAGEN) and analyzed by qRT-PCR. For RIP in circ-ZNF609 knockdown, anti-rabbit-IgG and YTHDF1 polyclonal antibody (Proteintech, #17479-1-AP) or YTHDF2 polyclonal antibody (Proteintech, #24744-1-AP) were used. The RIP assay was conducted as the same procedure except AC16 cardiomyocytes were pretransfected with si-circ-ZNF609 or NC control.
9
Immunoprecipitation of HA-xCPEB4 Protein
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Stage VI oocytes were injected with 4.9 fmols of in vitro transcribed HA-xCPEB4 + 3’UTR (wild type or phospho-mutants) and collected at indicated times. Oocytes were homogenized with 10 μl/oocyte of IP lysis buffer (20 mM Tris-HCl pH 8, 1 mM EDTA, 0.5% NP-40, 1 mM MgCl2 and 100 mM NaCl) supplemented with H1 kinase buffer and EDTA-free protease inhibitors (Sigma-Aldrich). 280 μl of oocyte extract was pre-cleared with 25 μl of Dynabeads protein G (Invitrogen) and then incubated for 2 hr at 4°C with anti-HA antibody covalently cross-linked to Dynabeads protein G. Immunoprecipitates were washed six times with IP lysis buffer and eluted with Laemmli sample buffer by heating at 65°C for 20 min. Eluates were resolved in SDS-PAGE, and analysed by Western blotting, silver staining (Pierce Silver Stain for Mass Spectrometry, Thermo Fisher Scientific) or colloidal blue staining (Invitrogen). Mass spectrometry analysis was performed at the Mass Spectrometry Core Facility at IRB Barcelona, as described previously.
10
Protein-protein interaction assay
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A total of 5 μg mouse anti-His, anti-HA, or anti-Flag monoclonal antibody (CWBiotech) was incubated with 50 μL Dynabeads Protein G (Novex, Thermo Fisher Scientific) for 15 min at room temperature. Then, 400 µL 1:1 mixture of recombinantly expressed importin α2 fragment (IBB-His, Arm-His or carboxyl-terminal–His) and NP-Flag, importin β–HA and NP-Flag, importin β–HA and IBB-His, or HSPG His-tagged five fragments and NP-Flag were added and incubated for 2 h at 4 °C. In another group, 400 µL IBB-His and 400 µL total protein from viruliferous planthoppers in 10 mM PBS buffer (pH 8.0) was added and incubated for 2 h at 4 °C. The total protein from E. coli–expressing empty pET28a was applied in the negative control groups. A total of 5 μg NP monoclonal antibody was first incubated with 50 μL Dynabeads Protein G (Novex) for 30 min at room temperature, after which 400 μL total protein from viruliferous planthoppers in 10 mM PBS buffer (pH 8.0) was added. Approximately 10% of the total protein was reserved as input. Mouse IgG (Merck Millipore) was used as a negative control. After washing three times with washing buffer (Novex), the antibody–protein complex was disassociated from the beads with elution buffer (Novex) for Western blot analysis with anti-His, anti-HA, anti-Flag, or anti-NP monoclonal antibodies or anti–importin α2 polyclonal antibodies.
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