Cells or homogenized tissues were lysed in lysis buffer (50 mM HEPES-NaOH, pH 7.5, 20 mM MgCl2, 150 mM NaCl, 1 mM dithiothreitol [DTT], 1 mM phenylmethane sulfonylfluoride [PMSF], 1 μg/ml leupeptin, 1 mM EDTA, 1 mM Na3VO4, 10 mM NaF, 0.5% NP-40, 1% 3-((3-cholamidopropyl)dimethylammonium)-1-propanesulfonate [CHAPS], and 0.3% SDS) and centrifuged as previously described (Yamauchi et al., 2012 (link); Miyamoto et al., 2013b (link)). The supernatants were mixed with protein G resin that was preadsorbed with an anti–cytohesin-2 antibody. The immunoprecipitates or proteins in the lysates were denatured and then separated on SDS–polyacrylamide gels. The electrophoretically separated proteins were transferred to polyvinylidene difluoride (PVDF) membranes, blocked with the Blocking One kit (Nacalai Tesque), and immunoblotted first with primary antibodies and then with peroxidase-conjugated secondary antibodies. The bound antibodies were detected using the Chemi-Lumi One L kit (Nacalai Tesque). The band images were captured with a GT-7300U scanner (Epson, Tokyo, Japan) and analyzed with ImageJ software. At least three separate experiments were carried out under each condition, and a representative blot is shown in each of the figures.
Chemi lumi one l kit
Manufactured by Nacalai Tesque
Sourced in Japan
The Chemi-Lumi One L kit is a laboratory equipment designed for chemiluminescence detection and analysis. It provides a platform for researchers to perform sensitive and quantitative measurements of light-emitting chemical reactions.
Automatically generated - may contain errors
Lab products found in correlation
Imagequant las 4000 mini biomolecular imager, by GE Healthcare (3 mentions)
Immobilon p membrane, by Merck Group (2 mentions)
Imagequant tl software, by GE Healthcare (1 mentions)
Anti pcna antibody, by Santa Cruz Biotechnology (1 mentions)
Ab17984, by Abcam (1 mentions)
Anti ubiquitin monoclonal antibody, by Santa Cruz Biotechnology (1 mentions)
Anti pcna, by Santa Cruz Biotechnology (1 mentions)
Anti usp7, by Abcam (1 mentions)
Anti ubiquitin, by Santa Cruz Biotechnology (1 mentions)
Edta free protease inhibitor cocktail, by Nacalai Tesque (1 mentions)
Protease inhibitor mixture, by Nacalai Tesque (1 mentions)
Las 3000 bioimaging system, by Fujifilm (1 mentions)
Imagequant las 4000, by GE Healthcare (1 mentions)
Immobilon p, by Merck Group (1 mentions)
8 protocols using chemi lumi one l kit
1
Immunoprecipitation and Western Blot Analysis
2
PCNA Ubiquitination Assay Protocol
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PCNA-ubiquitination assays were performed as described (21 (link)). Briefly, the reaction mixture (25 μl) contained 20 mM HEPES-NaOH (pH 7.5), 50 mM NaCl, 0.2 mg/ml BSA, 1 mM DTT, 10 mM MgCl2, 1 mM ATP, poly(dA)-oligo(dT) (GE Healthcare) (100 ng), PCNA (1.0 pmol trimer), E1 (0.85 pmol), RAD6A-(His-RAD18)2 (0.54 pmol trimer), Ub (174 pmol) and DNA polymerases (2.5 pmol unless indicated otherwise). Reaction mixtures were prepared on ice, and then incubated at 30°C for 30 min unless indicated otherwise. The reactions were terminated with sample buffer for SDS-PAGE. Products were analysed by western blotting with anti-PCNA antibody (Santa Cruz Biotechnology, sc-7907). Signals were detected with a Chemi-Lumi One L kit (Nacalai Tesque, 07880–70) using ImageQuant™ LAS 4000 Mini Biomolecular Imager (GE Healthcare), and analysed using ImageQuant™ TL software (GE Healthcare).
3
Ubiquitin and PCNA Regulation Assay
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Reaction products were analyzed by western blotting. Anti-ubiquitin monoclonal antibody (Santa Cruz Biotechnology, Ub [P4D1] sc-8017), anti-PCNA monoclonal antibody (Santa Cruz Biotechnology, PCNA [PC10] sc-56), anti-RFC1 polyclonal antibody (Santa Cruz Biotechnology, RFC1 [H300] sc-20993), anti-HLTF monoclonal antibody (Abcam, [EPR14761] ab183042), and anti-HLTF serum raised in a rabbit against a histidine-tagged N-terminal fragment of HLTF (1–148 amino acids) were used. In co-immunoprecipitation assays, proteins were detected using anti-RFC1 polyclonal antibody (Abcam, ab3556), anti-HLTF polyclonal antibody (Abcam, ab17984), and anti-FLAG M2 monoclonal antibody (SIGMA-ALDRICH, F1804). Proteins were separated on SDS 4–20% gradient or 15% polyacrylamide gels, blotted onto a PVDF membrane, and probed with the indicated antibodies. Signals were detected with a Chemi-Lumi One L kit (Nacalai Tesque, 07880-70) using the ImageQuant™ LAS 4000 Mini Biomolecular Imager (GE Healthcare) and analyzed using ImageJ 1.48v software (NIH).
4
Antibody Detection and Imaging Protocol
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Anti-E6AP anti-serum was raised in a rabbit against the peptide KKGPRVDPLETELGVKTLDC. Antibodies used included anti-PCNA (Santa Cruz Biotechnology, PC10, sc-56), anti-p53 (Calbiochem, Ab-6 mouse mAb (DO-1), OP43), anti-UBCH5c (Ab Frontier (4C1-1E3), LF-MA10362), anti-USP7 (Abcam, ab4080), and anti-ubiquitin (Santa Cruz Biotechnology, P4D1, sc-8017). Signals were detected with a Chemi-Lumi One L kit (Nacalai Tesque, 07880-70) using ImageQuant™ LAS4000 Mini Biomolecular Imager (GE Healthcare), and analyzed using ImageJ 1.48v software (National Institutes of Health).
5
Affinity Purification of mCherry-IFT54
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IFT54-KO cells stably expressing an mCherry-fused IFT54 construct were grown to almost confluency on a 15-cm plate. The cells were lysed in 1 mL of lysis buffer (20 mM HEPES-KOH [pH 7.4], 3 mM MgCl2, 1 mM dithiothreitol, 100 mM KCl, 10% glycerol, 5 mM NaCl, and 0.1% Triton X-100) containing EDTA-free protease inhibitor cocktail (Nacalai Tesque) by placing on ice for 40 min. After centrifugation of the lysates at 16,000×g at 4°C for 20 min, the supernatants were incubated with 40 µl of GST-tagged anti-mCherry Nb prebound to glutathione-Sepharose 4B beads. The beads were washed five times with the lysis buffer and boiled in SDS-PAGE sample buffer. The proteins bound to the beads were then separated by SDS-PAGE, and electroblotted onto an Immobilon-P membrane (Merck Millipore). The membrane was blocked in 5% skimmed milk and incubated sequentially with the primary antibody and the peroxidase-conjugated secondary antibody. Protein bands were detected with a Chemi-Lumi one L kit, Chemi-Lumi super kit or Chemi-Lumi ultra kit (Nacalai Tesque).
6
Immunoblot Analysis of Cellular Proteins
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Immunoblot analysis was performed as described previously (Katoh et al., 2015 (link), 2016 (link)). Proteins in cell lysates prepared as described or on beads after the VIP assay were separated by SDS–PAGE and electroblotted onto an Immobilon-P membrane (EMD Millipore). Membranes were blocked in 5% skim milk and incubated sequentially with a primary antibody (anti-GFP, anti-RFP antibody, anti-HA, or anti-IFT139) and horseradish peroxidase–conjugated secondary antibody. Detection was carried out using the Chemi-Lumi One L Kit (Nacalai Tesque).
7
Immunoblot Analysis of Protein Lysates
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Cells were lysed in lysis buffer (20 mM HEPES, pH 7.4, 150 mM NaCl, 1% Nonidet P-40, 1 mM EDTA) containing a protease inhibitor mixture (Nacalai Tesque) at 4°C for 30 min. The lysates were centrifuged at maximum speed for 20 min at 4°C in a microcentrifuge to remove cellular debris and insoluble materials. Cell lysates (10 μg/sample) were incubated in SDS sample buffer containing β-mercaptoethanol at 37°C for 2 h or at room temperature overnight, and then the samples were subjected to SDS–PAGE and immunoblot analysis using rat anti-HA, mouse anti-actin, or mouse anti-TfnR antibody. Immunoblots were developed using a Chemi-Lumi One L kit (Nacalai Tesque), and recorded on a LAS-3000 bioimaging system (Fujifilm).
8
Co-Immunoprecipitation and Immunoblotting
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The Co-IP supernatants (10 μL) were subjected to SDS-PAGE and transferred onto a polyvinylidene difluoride (PVDF) membrane (Immobilon P; EMD Millipore Corporation, Billerica, MA, USA) with Towbin buffer (40 mM Tris-HCl, 38 mM glycine, 10% methanol, 0.025% SDS). Similarly, the cell lysates (10 μg of intracellular proteins) were simultaneously subjected to SDS-PAGE and immunoblotted (IB) for references. The PVDF membranes were then blocked in 0.3% skim milk in PBS containing 0.1% Tween 20 (PBST) and probed with the antibodies against DYKDDDDK and c-Myc (Wako Pure Chemical Industries Ltd.). TrueBlot ® Anti-IgG HRP (Rockland Immunochemicals Inc., Limerick, PA, USA) was used for the detection of immunoblotted target protein bands. The chemiluminescence signal was detected by using the Chemi-Lumi One L Kit (NacalaiTesque, Kyoto, Japan) along with the ImageQuant LAS-4000 image analyzer (GE Healthcare, Piscataway, NJ, USA).
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