Cells were transfected with the indicated plasmids and lysed in IP lysis buffer 48 h after transfection. Following lysis, protein estimation was done using BCA Kit (Pierce USA). 100 µg lysate was taken and cleared out using Protein A/G agarose beads (Sigma Aldrich) by incubating them in a spin rotator for 1 h., following which lysate was centrifuged to remove the Protein A/G agarose beads. About 1 μg of the indicated antibody i.e., anti-USP37 (Novus Biologicals, #cat NB110-40709) or Anti PCNA Antibody, was mixed with lysate at 4 °C overnight. Protein A/G beads were then added and incubated at 4 °C for 1 h. to capture the antibody-antigen complex. Beads were washed three times with IP lysis buffer, and proteins were eluted by boiling beads in Laemmli buffer for 5 min and visualized by immunoblotting using respective antibodies as indicated.
Protein a g agarose beads
Manufactured by Merck Group
Sourced in United States, Germany
Protein A/G agarose beads are a type of chromatography resin used for the purification of antibodies. The beads are composed of agarose, a polysaccharide derived from seaweed, and covalently linked with either Protein A or Protein G, which are bacterial proteins that have a high affinity for the Fc region of immunoglobulins. These beads can be used to selectively capture and purify antibodies from complex mixtures, such as cell culture supernatants or serum samples.
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Lab products found in correlation
Protease inhibitor cocktail, by Roche (7 mentions)
Anti flag, by Merck Group (4 mentions)
Protein a g agarose bead, by Santa Cruz Biotechnology (4 mentions)
Protease inhibitor cocktail, by Merck Group (4 mentions)
Direct zol rna miniprep kit, by Zymo Research (4 mentions)
Sonifier 250, by Emerson (3 mentions)
Genome analyzer 2, by Illumina (3 mentions)
Anti bax antibody, by Santa Cruz Biotechnology (2 mentions)
Radioimmunoprecipitation assay buffer, by Merck Group (2 mentions)
Anti bcl 2, by Santa Cruz Biotechnology (2 mentions)
Anti phospho bad, by Cell Signaling Technology (2 mentions)
Anti bad, by Santa Cruz Biotechnology (2 mentions)
Anti 14 3 3, by Santa Cruz Biotechnology (2 mentions)
Anti bcl xl antibody, by Santa Cruz Biotechnology (2 mentions)
N ethylmaleimide, by Merck Group (2 mentions)
Hiseq 2000, by Illumina (2 mentions)
Igepal ca 630, by Merck Group (2 mentions)
Normal rabbit igg, by Santa Cruz Biotechnology (2 mentions)
Western and ip lysis buffer, by Beyotime (2 mentions)
Phosphatase inhibitor, by Beyotime (2 mentions)
93 protocols using protein a g agarose beads
1
Immunoprecipitation of USP37 and PCNA
2
Investigating NCOA3 Interactions in HCC Cells
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To investigate the interaction between NCOA3 and transcription factors in HCC cells, Co-IP assay was performed. Totally, 5 × 106 HCC cells were harvested, washed three times with cold PBS, and treated with Hypotonic Buffer (20 mM Tris-HCl, pH 7.4, 10 mM NaCl, 3 mM MgCl2·6H2O) for 15 min on ice. Totally, 10% NP40 was added and vortexed for 15 s. The cells were pelleted and treated with cell extraction buffer (100 mM Tris, pH 7.4, 2 mM Na3VO4, 100 mM NaCl, 1% Triton X-100, 1 mM EDTA, 10% glycerol, 1 mM EGTA, 0.1% SDS, 1 mM NaF, 0.5% deoxycholate, 20 mM Na4P2O7·10H2O) supplemented with cocktail protease inhibitors in ice for 30 min. After centrifugation, the supernatant was collected and pre-cleared with 50% protein A/G agarose beads (Millipore, Billerica, MA) for 1 h. Primary antibody was then added in for incubation overnight, and new protein A/G agarose beads were used to pull down proteins interacting with NCOA3.
3
Immunoprecipitation of Mfsd2a and ZIKV E
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For immunoprecipitation of ectopically expressed Mfsd2a and ZIKV E, HEK293T cells in 100-mm plates were transfected with Mfsd2a-Myc plasmids, followed by Flag-tagged ZIKV E transfection 12 hours later. At 24 hours after transfection, MG132 was added, and a further 6-hour incubation was allowed before lysing the cells. For immunoprecipitation of the ZIKV E protein with endogenous Mfsd2a, Flag-tagged ZIKV E or Flag-tagged WNV E plasmids were transiently transfected into HUVECs in the presence or absence of MG132. The lysate was cleared by centrifugation at 14,000g for 20 min at 4°C and incubated for 1 hours at 4°C with 4 μg of M2 anti-Flag antibody (Sigma-Aldrich). Then, the immune complex was coupled to Protein A/G agarose beads (Sigma-Aldrich) overnight at 4°C. The beads were washed with lysis buffer five times, and samples were boiled in 2× SDS loading buffer, resolved by 10% SDS-PAGE, and then probed with monoclonal antibodies against c-Myc, Mfsd2a, or Flag, respectively. Five percent of the inputs were detected with corresponding antibodies to ensure the expression of transfected genes.
4
Flag/HA Immunoprecipitation Assay
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293T cells were collected and lysed in RIPA lysis buffer (Yeasen, Shanghai, China) supplemented with protease inhibitor Cocktail (Yeasen) for 30 min at 4 °C, centrifuged at 8800 rpm for 10 min, the supernatant was collected and divided into three 1.5 mL tube (one for input, one for IgG, and one for IP). For the immunoprecipitation assay, the cell lysate supernatants were incubated for 4 h at 4 °C with anti-Flag (Sigma) or anti-HA (Cell Signaling) coupled to the protein A/G-agarose beads (Sigma). Then, immunoprecipitated beads were washed 5–6 times with RIPA lysis buffer. Immunoprecipitates and input were subjected to run SDS-PAGE gel, and the next step was the same as the immunoblot assay.
5
Quantification of Protein Acetylation
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Whole-cell lysate prepared as described [31 (link)] was used to determine the levels of proteins. Antibodies used in this study include those against XPA (Kamiya), XPB-XPD (Santa Cruz Biotechnology, Santa Cruz, CA, USA), XPE, p-p53, p-CHK1, GAPDH, SIRT1, acetyl-lysine (Cell Signaling Technology), XPF, and XPG (both Abcam, Cambridge, UK). For immunoprecipitation of XPA, 1 mg of whole-cell lysate was incubated with 1 μg of anti-XPA conjugated to Protein A/G-agarose beads (Sigma, St. Louis, MO, USA) for 12 h at 4°C with rotation. After washing with lysis buffer, proteins were eluted from the beads by boiling in SDS sample buffer and resolved on 10% SDS-polyacrylamide gels. For detection of XPA acetylation, anti-acetyl-lysine was employed.
6
Quantifying 35 K-Fc in Plasma
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35 K-Fc was detected in plasma by immunoprecipitation with Protein A/G agarose beads (Sigma, UK). Plasma samples were diluted 1:2 with Cell-lytic MT lysis buffer (Sigma, UK) and incubated with Protein A/G beads overnight at 4 °C with rotational mixing. The beads were washed 5 times with the lysis buffer prior to solubilisation of the bound proteins by LDS containing SDS-PAGE loading buffer (Invitrogen, UK). The resulting protein fraction was run on a western blot on a Nu-Page gel (Invitrogen) and transferred onto PVDF membrane (Amersham). The proteins were detected by blotting with anti-35 K antibodies (R&D Systems) and anti-human Fc (Jackson Labs, US) antibodies.
7
ChIP Analysis of Chromatin Modifications
8
Immunoprecipitation of Phospho-Bad Interactions
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Immunoprecipitation was performed to examine the interaction of phospho-Bad and 14-3-3 interaction and Bcl-2 family proteins interaction. Protein samples (200 μg) were precleared with protein A/G agarose beads (Santa Cruz Biotechnology) to eliminate nonspecific binding proteins. They were mixed with following antibodies: anti-14-3-3, anti-Bad, and anti-Bax antibody (Santa Cruz Biotechnology) and incubated for overnight at 4°C with mild shaking. Complexes were precipitated with protein A/G agarose beads for 2 h at 4°C, washed with radioimmunoprecipitation assay buffer (Sigma Aldrich) with PMSF, and centrifuged at 10,000 g for 1 min. Supernatants were removed and remnant were boiled with sample buffer. They were loaded on 10% sodium dodecyl sulfate polyacrylamide gel, electrophoresed, and transferred to PVDF membrane. Membrane were rinsed with TBST and reacted with following antibodies: anti-phospho-Bad (1:1,000, diluted with TBST, Cell Signaling Technology), anti-Bcl-2, and anti-Bcl-xL antibody (1:1,000, diluted with TBST, Santa Cruz Biotechnology). They were washed with TBST and continuously performed as above described method in Western blot analysis.
9
Immunoprecipitation and Western Blot Analysis
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Cells were transfected as aforementioned and grown in 10-cm dishes. To extract proteins, cells were washed with ice-cold PBS and lysed in NETN lysis buffer (150 mM NaCl, 1 mM EDTA, 20 mM Tris-Cl pH 8.0, 0.5%NP-40(v/v) containing protease and phosphatase inhibitors (50 mM NaF, 0.1 mM Na3VO4). The lysates were incubated, respectively, with 2 μg of IP antibodies to Myc (9E10) (#05419, Millipore) or Axl (C-20) (#sc-1096, Santa Cruz) at 4 °C overnight with constant rotation. The immunocomplex was captured by incubating with 40 μl protein A/G-agarose beads (Sigma Aldrich, Sr. Louis, MO, USA) for 3 h at 4 °C with constant rotation. Following 1 min centrifugation at 10000 g, the beads were washed three times with ice-cold NETN buffer and ready for SDS-PAGE analysis or kinase assay. Representative Western blot results are shown in figures and experiments were performed in triplicate.
10
Detecting Oxidized Cysteines in CSE Protein
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Oxidized cysteines in the CSE protein were detected with a Co-IP assay, as previously described [34 (link)]. Briefly, the proteins (120 µg) were first incubated with 1 μg of anti-cysteine sulfonate antibody (Abcam) overnight at 4 °C, followed by incubation with protein A/G-agarose beads (Sigma-Aldrich) for 2 h at 4 °C. The beads were washed three times with lysis buffer, and the bound proteins were then analyzed by Western blotting with an anti-CSE antibody. The total proteins that had not undergone the Co-IP procedure were assessed for the detection of GAPDH expression with anti-GAPDH antibody. The cysteine sulfonate from the CSE protein was normalized to the GAPDH protein level.
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