For immunoprecipitation of RNA, two rounds using 5 μg of anti-m6A antibody and 4 μg of small RNA were performed. The reaction was carried out using the Immunoprecipitation Kit—Dynabeads Protein G with some modifications (Life Technologies, Carlsbad, CA, USA)[4 (link)]. First, the antibody was coupled to Dynabeads Protein G in 500 μl of Binding and Washing Solution for 3 hours at 4°C followed by 10 minutes incubation at room temperature. Beads were then washed three times in Washing Buffer. Small RNA was added to the antibody-coupled beads in IP buffer (140 mM NaCl, 10 nM sodium phosphate, 0.05% Triton-X) supplemented with RNase inhibitor—Superase-In (Life Technologies, Carlsbad, CA, USA) and kept on the rotating platform at 4°C overnight. On the next morning beads were washed 5 times with IP buffer. Finally beads were treated with 250 μl of Elution Buffer (5 mM Tris pH 7.5, 1 mM EDTA, 0.05% SDS) supplemented with 2.1 μl Proteinase K (Invitrogen, 20 mg/ml) for 1.5 h on a heating block at 50°C at 1100 rpm. Immunoprecipitated RNA was recovered with Trizol LS reagent following the instructions from the manufacturer. As a control, immunoprecipitation was performed using IgG instead of anti-m6A antibody. The rest of experimental parameters were kept identical.
Immunoprecipitation kit
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Immunoprecipitation kit is a laboratory tool designed to isolate and purify specific proteins from complex mixtures, such as cell lysates or tissue extracts. The kit includes all the necessary components to perform this process, including antibodies, beads, and buffers.
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Lab products found in correlation
Pierce bca protein assay kit, by Thermo Fisher Scientific (4 mentions)
Dynabeads protein g, by Thermo Fisher Scientific (3 mentions)
Dynabeads streptavidin trial kit, by Thermo Fisher Scientific (2 mentions)
Hur antibody or mouse igg, by BD (2 mentions)
Random rna oligo, by Horizon Discovery (2 mentions)
Unbiotinylated bsg oligo, by Horizon Discovery (2 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions)
Immunoprecipitation kit, by Promega (2 mentions)
Recombinant mouse il18, by Thermo Fisher Scientific (2 mentions)
Il18 polyclonal antibody, by Abcam (2 mentions)
Protein a agarose, by Thermo Fisher Scientific (2 mentions)
Superase in rnase inhibitor, by Thermo Fisher Scientific (1 mentions)
Proteinase k, by Thermo Fisher Scientific (1 mentions)
Anti rac1, by Merck Group (1 mentions)
Anti stat3, by Cell Signaling Technology (1 mentions)
Finnigan lcq deca iontrap mass spectrometer, by Thermo Fisher Scientific (1 mentions)
High performance liquid chromatography system, by Agilent Technologies (1 mentions)
Protease inhibitor cocktail tablet, by Roche (1 mentions)
Amicon ultra 0.5 centrifugal filter unit, by Merck Group (1 mentions)
Dynabeads m 280 sheep anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
32 protocols using immunoprecipitation kit
1
RNA Immunoprecipitation with m6A Antibody
2
Investigating Protein Interactions by Co-IP
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Co-immunoprecipitation (Co-IP) experiments were performed on whole ovary protein extracts obtained from 40 ovaries using an immunoprecipitation kit (Life Technologies) according to the manufacturer’s instructions. Antibodies used for IP were anti-Rac1 (Millipore), anti-STAT3 (Cell Signaling), normal rabbit serum, or normal mouse serum (IgG). The precipitated protein complexes were then used for Western blot analysis as described above.
3
Immunoprecipitation and Mass Spectrometry of GIT Protein
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Whole brains were excised from 8-week-old mice, homogenized in IP buffer (250 mM hydroxyethyl piperazine ethanesulfonic acid (pH 7.4), 100 mM NaCl, 10 mM KCl, 10 mM NaF, 1 mM Na3VO4, 10 mM β-glycerophosphate, 10% glycerine, 0.5% CHAPS and 0.5% Triton X-100) supplemented with protease inhibitor cocktail tablets (Roche, Shanghai, China), and centrifuged for 20 minutes at 12,000 × g at 4°C The supernatant, containing 1 mg total protein, was incubated with 10 μg anti-GIT antibody (Santa Cruz Biotechnology). The target antibody complex was immunoprecipitated with Dynabeads® Protein G (Life Technologies) and eluted with elution buffer in the immunoprecipitation kit (Life Technologies). The eluted supernatant was subjected to polyacrylamide gel electrophoresis (PAGE) for mass spectrometric detection or protein immunoblotting. For mass spectrometry, the proteins were excised from the stacking gel and digested with trypsin as previously described (Koles et al., 2012). The peptides were separated using a high performance liquid chromatography system (Agilent, Palo Alto, CA, USA) and detected with a ThermoElectron Finnigan LCQ DECA ion trap mass spectrometer (Thermo).
4
Nuclear Protein Extraction and Analysis
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Nuclear extracts (NE) from RCC FG1 cells and HeLa cells were prepared with NE-PER nuclear and cytoplasmic extraction reagents-kit obtained from Thermo Scientific, Dreieich, Germany. The buffer was changed to RIPA buffer (50 mM Tris–HCl pH 7.4, 150 mM NaCl, 1 mM EDTA, 1 % Triton X-100, 1 % sodium deoxycholate, 0.1 % SDS) by using Amicon Ultra-0.5 centrifugal filter units with ultracell-10 membrane (Merck Millipore, Schwalbach, Germany). For co-immunoprecipitation the Dynabeads M-280 sheep anti-rabbit IgG and the immunoprecipitation kit purchased from life technologies was used. NE were incubated with antibody-coupled Dynabeads for 20 min at room temperature. Samples were analyzed by SDS-Page and Western Blot. Blue native polyacrylamide gel electrophoresis (BN-PAGE) was performed essentially as described by Fiala and Blumenthal [21 (link)]. NE from HeLa cells were incubated in native sample buffer (50 mM BisTris, 125 mM 6-AcA, 0.1 % Triton X-100, pH 7.0). After 10 min 0.5 % coomassie brilliant blue G250 was added and incubated for 5 min at 4 °C. The samples were separated on 20 to 8 % native gradient gels. Second dimension was separated by 10 % SDS-PAGE and then transferred on a PVDF membrane for immunoblot.
5
Co-immunoprecipitation of BATF2 Complexes
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Co‐immunoprecipitation (co‐IP) was performed by using an immunoprecipitation kit (Novex, Life Technologies, Carlsbad, CA USA). Briefly, immunoglobulin G (IgG) or BATF2 antibody (2 µg) was incubated with Dynabeads protein G with rotation for 1 h and immunoprecipitated with 500 µg protein at 4°C overnight. After elution with elution buffer, the supernatant was collected for Western blot or proteomic analysis. IgG served as negative control, while total protein served as the input.
6
T Cell Activation and Apoptosis Assay
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TCRα and Fas shRNA reagent kits (sc-37273-SH), antibodies of CD3 (Alexa Fluor® 488; PC3/188A), CD4 (Alexa Fluor® 546; MT310), IL-4 (Alexa Fluor® 594; OX81), CD154 (Alexa Fluor® 647; F-1), CD9 (C-4), CD63 (MX-49.129.5), CD81 (B-11), MHC II (Y-Ae), Ovalbumin (2D-11), Fas (G-9), and TCR (R73) were purchased from Santa Cruz Biotech (Santa Cruz, CA). Biotinylated anti-CD9 Ab and anti-biotin magnetic micro beads were purchased from Miltenyi Biotech (San Diego, CA). Casp3 protein, ELISA kits of Casp3, IgE, mMCP1, IL-4, IL-5 and IL-13 were purchased from R&D Systems (Minneapolis, MN). Ovalbumin, FITC-annexin V kit and FITC-dextran were purchased from Sigma Aldrich (St. Louis., MO). Immunoprecipitation kit and materials for Western blotting were purchased from Invitrogen (Carlsbad, CA). Magnetic beads coated with anti-CD9 antibody was purchased from AMS Biotechnology (Abingdon, UK).
7
Protein-Protein Interaction Assay for SARS-CoV-2 RBD
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Protein pull-down assay was performed using Immunoprecipitation kit (Invitrogen) as previously described (20 (link)). Briefly, 10 μl protein A beads were incubated with 1 μg SARS-CoV-2 RBD-Fc at room temperature for 1 hour. Then different amounts (7.04, 3.52. 1.76, 0.88, 0.44, 0.22, or 0 μg) of Nanosota-1C (with a C-terminal His tag) and 4 μg human ACE2 (with a C-terminal His tag) were added to the RBD-bound beads. After one-hour incubation at room temperature, the bound proteins were eluted using elution buffer (0.1 M glycine pH 2.7). The samples were then subjected to SDS-PAGE and analyzed through Western blot using an anti-His antibody.
8
Co-Immunoprecipitation and Immunoblotting
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CO-IP was performed using an immunoprecipitation kit (Invitrogen) according to the manufacturer’s recommended protocol. Then, the cell lysates and immunoprecipitates were analyzed by immunoblotting. The specific antibodies for CO-IP and immunoblotting are provided in Additional file
8 .
9
C/EBPβ and RUNX2 Protein Interaction
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Nuclear protein was extracted from ATDC5 cells that had been transfected with the C/EBPβ expression vector. IP was performed with an Immunoprecipitation kit (Invitrogen) according to the manufacturer's instructions. For immunoprecipitation, nuclear extract was incubated with magnetic beads conjugated with C/EBPβ, RUNX2 or normal rabbit IgG antibody for 10 minutes. Analysis was performed by immunoblotting.
10
Analyzing PGC-1α Acetylation in Heart Tissue
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Whole hearts were homogenized in 500 μl ice-cold lysis buffer (25 mmol/L Tris HCl, ph 7.4, 5 mmol/L MgCl2, 10% glycerol, 100 mmol/L KCl, 1% NP40, 0.3 mmol/L dithiothreitol, 5μl protease/phosphatase inhibitor cocktail, 5 mmol/L nicotinamide, 1 μmol/L orthovanadate, 50 mmol/L sodium fluoride, 1 mmol/L sodium butyrate and 5 mmol/L sodium pyrophosphate), sonicated for 1 min using an Ultra-Turrax T10 basic (IKA Labortechnik, Staufen, Germany), and separated by centrifugation (10 000 x g for 30 min at 4°C). 500 μg of protein were incubated with 50 μl beads (1.5 mg) and 5 μg anti-PGC-1α antibody (Santa Cruz, Heidelberg, Germany), rotating overnight at 4°C. Beads were collected using a magnetic rack, washed with washing-buffer (Immunoprecipitation kit, Invitrogen, Carlsbad, CA), dissolved in loading buffer, and boiled. The immunoprecipitates were then separated by SDS-PAGE and immunoblotted using anti-PGC-1α-antibody (1:200; Santa Cruz, Heidelberg, Germany) and subsequently with acetyl-lysine antibody (1:1.000; Cell Signaling, Boston, MA).
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