For the endogenous IP assay, bladder cancer cells were directly collected and subjected to the following protocols. For the exogenous IP assay, the cells were first transfected with the overexpressing YAP1 plasmid vector with flag-tag (YAP1-flag-tag; GenePharma), and were then collected for the following protocols. In detail, bladder cancer cells were first rinsed with cold PBS and lysed in IP lysis buffer (Thermo Fisher Scientific, Inc.), and the total protein in the lysate served as the 'Input' sample. Then, cell lysate containing 200 µg protein was incubated with Dynabeads® Protein G (Thermo Fisher Scientific, Inc.) for 1 h, and incubated with 2 µg antibody against YAP1 (cat. no. PA5-78321, Invitrogen; Thermo Fisher Scientific, Inc.), mTOR (cat. no. PA5-34663, Invitrogen; Thermo Fisher Scientific, Inc.) flag (cat. no. 8146, Cell Signaling Technology, Inc.), or beads (negative control) overnight at 4°C, followed by incubation with Dynabeads® Protein G for another 1 h to form the immune complex, which was considered as the 'Elute' sample. Subsequently, both the 'Input' and 'Elute' samples were loaded onto gels for western blotting with antibodies against Ub (cat. no. 3933, Cell Signaling Technology, Inc.), SKP2 (cat. no. ab68455, Abcam) or mTOR (cat. no. PA5-34663, Invitrogen; Thermo Fisher Scientific, Inc.).
Dynabeads protein g
Manufactured by Cell Signaling Technology
Sourced in United States
Dynabeads® Protein G are uniform, superparamagnetic beads coated with recombinant Protein G. Protein G is a bacterial-derived protein that binds to the Fc region of immunoglobulins from various species. The beads enable the rapid and efficient capture and isolation of antibodies from complex samples.
Automatically generated - may contain errors
Lab products found in correlation
Ip lysis buffer, by Thermo Fisher Scientific (3 mentions)
Dynabeads protein g, by Thermo Fisher Scientific (2 mentions)
Protein g dynabead, by Thermo Fisher Scientific (2 mentions)
Protease inhibitor, by Roche (1 mentions)
Power sybr green pcr master mix, by Thermo Fisher Scientific (1 mentions)
Anti phospho tyrosine, by Cell Signaling Technology (1 mentions)
Dynabeads protein g, by Abcam (1 mentions)
Anti pak1, by Cell Signaling Technology (1 mentions)
Myc tag 9b11 mouse mab, by Cell Signaling Technology (1 mentions)
Complete mini, by Roche (1 mentions)
Anti gata2, by Cell Signaling Technology (1 mentions)
Ne per nuclear and cytoplasmic extraction kit, by Thermo Fisher Scientific (1 mentions)
Stat3 antibody, by Cell Signaling Technology (1 mentions)
Mccoy s 5a modified medium, by Thermo Fisher Scientific (1 mentions)
Nupage lds sample buffer, by Thermo Fisher Scientific (1 mentions)
Nupage sample reducing agent, by Thermo Fisher Scientific (1 mentions)
Image lab, by Bio-Rad (1 mentions)
Control igg, by Merck Group (1 mentions)
C jun, by Cell Signaling Technology (1 mentions)
12 protocols using dynabeads protein g
1
Endogenous and Exogenous IP Assay for Bladder Cancer
2
Chromatin Immunoprecipitation of TFEB
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The protocol was adopted from Blecher-Gonen et al. (2013) (link). Briefly, cells treated were fixed in 1% formaldehyde during 10 min. Glycine was then added to a final concentration of 0.15 M. Cells were washed with PBS and were lysed with ice-cold RIPA buffer supplemented with protease inhibitors (Roche). The chromatin fraction was sonicated to obtain fragments from 100 to 500 bp and were coupled with DynaBeads protein G coupled to the anti-TFEB antibody (D2O7D, Cell Signaling) overnight at 4°C. Immune complexes were eluted from the beads with 1% SDS in TE. After treatment with RNaseA and proteinase K, protein-DNA cross-links were reversed by adding NaCl 5M and incubated at 65°C overnight. Chromatin immunoprecipitation analysis was performed by qPCR using Power SYBR Green PCR Master Mix (Thermo Fisher). The primers for gla and mcoln1 were used from Palmieri et al. (2011) (link).
3
Detecting FOXM1-Cyclin D1 Interaction
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The IP-Kit–Dynabeads Protein G (Thermo Fisher Scientific, Waltham, MA) was used to detect the association of phospho-FOXM1 with cyclin D1. A 500 μg aliquot of total protein from cell lysate from the different conditions (see western blot analysis) was mixed with 500 μl of binding buffer. The 50 μl of Dynabeads® Protein G was incubated with 10 μg of cyclin D1 rabbit polyclonal antibody (#2922, Cell Signaling) for 30 min at RT. The Dynabeads-Ab complex was then resuspended with the cell lysate and incubated on a rotation rocker at RT for 2 h. The Dynabeads-Ab-Antigen complexes were eluted according to the manufacturer’s instructions, separated on SDS-PAGE, and analyzed by western blotting at the molecular weight of FOXM1 using rabbit polyclonal anti-phospho-tyrosine (#9411, Cell Signaling) and rabbit polyclonal anti-cyclin D1 antibodies.
4
Immunoprecipitation Assay for RAGE Protein
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An IP assay was performed using protein G plus A-agarose beads as established, with CDC4 or RAGE antibodies obtained from Santa Cruz Biotechnology, Inc. In brief, SW480 and T84 cells were first rinsed with cold PBS and lysed in IP lysis buffer (Thermo Fisher Scientific, Inc.); the total proteins were obtained after being centrifuged at 20,000 × g for 30 min at 4°C and served as the 'Input' sample. Then, the cell lysate containing 200 µg protein was incubated with Dynabeads® protein G (Thermo Fisher Scientific, Inc.) for 1 h at room temperature, and incubated with 2 µg of antibody against RAGE (cat. no. sc365154; Santa Cruz Biotechnology, Inc.) or IgG (cat. no. 5946, Cell Signaling Technology, Inc.; negative control) overnight at 4°C, followed by incubation with Dynabeads® protein G for another 1 h at room temperature to form the immune complex. Then, the protein samples were loaded onto gels for western blot analysis.
5
PTEN and Ubiquitin Protein Interaction
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The interaction between PTEN and Ub proteins was evaluated by IP assay. In brief, prostate cancer cells were washed with cold PBS and then lysed with IP lysis buffer (Thermo Fisher Scientific). Subsequently, cell lysate containing 200 μg proteins was incubated with Dynabeads® protein G for 1 h at room temperature, and cross-linked with 2 μg of antibody against PTEN (No. ab32199, Abcam) or Ub (No. 3936, Cell Signaling Technology, MA, USA) overnight at 4 °C. Next, the proteins were incubated with Dynabeads® protein G for another 1 h to form the immune complex which was loaded onto gels using anti-Ub (No. 3936, Cell Signaling Technology) or anti-PTEN (No. ab32199, Abcam) antibody.
6
PAK1 Immunoprecipitation Protocol
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Co-IP assays were performed using Dynabeads® Protein G to immunoprecipitate the lysates from OH931, NMFH1, and NMFH2 cell lines, with anti-PAK1 (Cell signaling) coupled to the beads with DMP (Sigma) as cross linkers. The procedures of co-IPs and dilution of antibodies in IPs and western blots are described in Method-S8.
7
Protein Extraction and Pulldown
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Cell lysates were harvested in mild Nonidet P-40 buffer with protease and phosphatase inhibitor as described above (50mM HEPES, 150 mM NaCl, 0.7% Nonidet P-40, 10% Glycerol, 1mM EDTA) with Roche cOmplete Mini protease inhibitor tablet and Roche PhoSTOP phosphatase inhibitor, at 0 °C. A total of 5 mg lysate in 1 mL total buffer was incubated with 30 μl MyOne Streptavidin C1 Dynabeads or 5 mg Myc-Tag (9B11) Mouse mAb (Cell Signaling, no. 2276) antibody for 3 h with subsequent 1-h incubation with 30 μl Protein G Dynabeads for streptavidin pulldown and myc-tag immunoprecipitation (IP), respectively. The beads were washed five times in lysis buffer before elution in NuPAGE 3-(N-morpholino)propanesulfonic acid (MOPS) sodium dodecyl sulfate (SDS) running buffer at 95 °C.
8
GATA2 ChIP-seq Protocol
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ChIP assays were performed as described previously [66 (link)]. Briefly, cells were fixed in 1% paraformaldehyde for 15 min, and chromatin was sheared to an average size of 300 bp by sonication. Lysates were incubated with 0.5 μg anti-GATA2 (Cell signaling technologies) or control rabbit IgG bound to Protein G Dyna beads (Cell Signaling technologies) overnight, subsequently washed with low- and high-salt buffers and eluted with 0.1 mol/L NaHCO3 in 1% SDS. Primer pairs are listed below.
9
Stat3 and Blcap Co-immunoprecipitation Protocol
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For Stat3 and Blcap co-immunoprecipitation, T24 bladder carcinoma cells were used. Cells were cultured in McCoy’s 5a modified medium (Invitrogen, Carlsbad, CA, USA) supplemented with 10% heat-inactivated FCS until they reached 70% confluency, they were then serum-starved for 1h and subsequently stimulated or not stimulated with IL-6 (20ng/ml) for 6 h to induce activation and nuclear translocation of Stat3. Cells were washed twice with ice-cold PBS and lysed with lysis buffer (25 mM Tris, 150 mM NaCl, 1 mM EDTA, 1% NP-40, 5% glycerol, pH 7.4) containing phosphatase and protease inhibitor mixtures (Roche Applied Science). For cell lysis and fractionation of cytoplasmic and nuclear protein extracts, the NE-PER Nuclear and Cytoplasmic Extraction Kit was used according to manufactorer’s instructions (Thermo Fischer Scientific, USA). For immunoprecipitation of proteins, lysates were precleared for 2h with Protein G-Dynabeads (Invitrogen) to minimize unspecific binding and then incubated overnight with a Stat3 antibody (Cell Signaling Technology, MA, USA) or isotype control pre-bound to Protein G-Dynabeads. The resin was then washed four times with ice-cold lysis buffer. Beads were resuspended in 30 μl of Laemmli sample buffer, boiled for 3 min and centrifuged at 14 000 g for 5 min. The supernatants were analysed by immunoblotting as described [1 (link)].
10
RNA-Binding Protein Immunoprecipitation Protocol
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Either 5 μg of anti‐PABPC1 antibody (10970‐1‐AP, Proteintech, Wuhan, China), anti‐YTHDF1 antibody (17479‐1‐AP, Proteintech, Wuhan, China) and IgG isotype control antibody (#3900S, Cell Signal Technology, USA) were conjugated to 100 μL of pre‐cleaned Protein G Dynabeads for overnight mixing at 4°C. Total protein of 10‐cm‐dish cultured cells was extracted using 400 μL IP Lysis buffer (10 mM Tris–HCl, 150 mM NaCl, 0.5% Igepal CA‐630). 10% Input samples were collected for further use. Antibody‐conjugated beads washed with twice 600 μL NT2 Wash Buffer (10 mM Tris–HCl, 150 mM NaCl, 2.5 mM MgCl2, 0.1% Igepal CA‐630), then mixed with total protein on a rotator at 4°C for 2 h. The beads were then sequentially washed with twice 600 μL NT2 wash buffers. 4× NuPAGE™ LDS Sample Buffer (NP0007, ThermoFisher, USA) and 10× NuPAGE™ Sample Reducing Agent (NP0009, ThermoFisher, USA) was added to 200 μL PBS dissolved beads, and the cells were heated at 70°C for 15 min. Then beads were then placed on magnetic stands and supernatant protein solution was moved to new EP tubes. Western blotting of 10% Input, IgG and IP was performed and expression levels were calculated using Bio‐Rad ImageLab version 6.1.
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