Cells were seeded in 10-cm dishes (2−8 × 104 cells/dish) and incubated for 24 h, after which inhibitors were added and cells were allowed to grow for the indicated periods of time. Cells were lysed in lysis buffer (25 mM HEPES, pH 7.6, 150 mM NaCl, 1% NP-40, 2% SDS, and 10% sucrose) supplemented with a protease inhibitor cocktail (Roche, Basel, Switzerland) and a phosphatase inhibitor cocktail (5 mM NaF, 200 μM sodium orthovanadate, 1 mM sodium molybdate, 2 mM sodium pyrophosphate, and 2 mM disodium beta-glycerophosphate). Protein concentrations were determined using OPA reagent (Thermo), according to the manufacturer’s instructions. Protein samples were separated by SDS-PAGE using gels prepared with WIDE RANGE Gel Preparation Buffer (Nacalai tesque, Kyoto, Japan). After electroblotting and blocking with skim milk, membranes were first reacted with a primary and then with appropriate secondary antibodies conjugated to fluorescent dyes. An Odyssey Infrared Imaging System (LI-COR Biosciences, Lincoln, NE, USA) was used for signal detection. The primary and secondary antibodies used are listed in Supplementary Table S1. ImageJ v1.49 (http://imagej.nih.gov/ij/ ) was used for image analysis.
Wide range gel preparation buffer
Manufactured by Nacalai Tesque
Sourced in Japan
WIDE RANGE Gel Preparation Buffer is a buffer solution used for preparing polyacrylamide gels in electrophoresis applications. It is designed to maintain a consistent pH range during the gel preparation process.
Automatically generated - may contain errors
Lab products found in correlation
Superose 6 pc 3.2 30 column, by GE Healthcare (2 mentions)
Smart system, by Cytiva (2 mentions)
Opa reagent, by Thermo Fisher Scientific (1 mentions)
Odyssey infrared imaging system, by LI COR (1 mentions)
Protease inhibitor cocktail, by Roche (1 mentions)
Phosphatase inhibitor cocktail, by Roche (1 mentions)
Ibright fl1500 imaging system, by Thermo Fisher Scientific (1 mentions)
Superdex 200 pc 3.2 30 column, by GE Healthcare (1 mentions)
5 protocols using wide range gel preparation buffer
1
Protein Analysis from Cell Lysates
2
Viral Protein Separation by SDS-PAGE
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Each of the prepared viruses was mixed with an equal volume of 2x SDS sample buffer (50 mM Tris-HCl, pH 6.5, 10% glycerol, 2% SDS and 0.1% bromophenol blue) and heated at 90°C. Samples were separated by electrophoresis on a 15% SDS-PAGE gel prepared with WIDE RANGE Gel Preparation Buffer (NACALAI TESQUE, Kyoto, Japan). After electrophoresis, the gel was stained with Coomassie brilliant blue and visualized with the use of the iBright FL1500 Imaging System (Thermo Fisher Scientific).
3
Gel-Filtration Chromatography of Recombinant Proteins
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Gel-filtration chromatography was performed using the SMART system (Amersham Pharmacia). Each purified recombinant protein and mixtures (each 2.4 μM) were incubated for 3 min at 60°C. Aliquots (20 μl) of each protein solution was applied to a Superose 6 PC 3.2/30 column (GE Healthcare) and eluted with buffer (50 mM Tris–HCl, pH 8.0 and 0.15 M NaCl). An aliquot (0.1 μl) of the protein solution and each eluted fraction (4 μl) were separated by 10% SDS-PAGE containing WIDE RANGE Gel Preparation Buffer (Nacalai Tesque), followed by silver staining. As an exception, in the experiments shown in Supplementary Figures S3 and S6 , proteins (each 4.5 μM) were mixed in the presence or absence of equimolar primed-DNA (annealed deoxyoligonucleotides d29 and d45, Supplementary Table S1 ). Each solution (25 μl) was applied to the column. An aliquot (5 μl) of the applied solution and each eluted fraction were separated by 10% SDS-PAGE, followed by CBB staining. Standard marker proteins thyroglobulin (670 000), γ-globulin (158 000), ovalbumin (44 000) and myoglobin (17 000) were run as controls.
4
CN-PAGE Analysis of ERp57-CNX Interaction
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CN-PAGE was performed using a modified version of the method of Pandhare et al. [28 (link)]. Purified wild-type ERp57 (2 μM) was incubated with CNX (2 μM) in buffer containing 50 mM Tris/HCl (pH 7.5), 0.05% (w/v) Coomassie Brilliant Blue (CBB) G-250, and 10% (v/v) glycerol in the absence or presence of 1 mM CaCl2 for 1 h at 30 °C to equilibrate intermolecular interactions. Samples were then incubated at 4 °C for 15 min, and separated by CN-PAGE or SDS-PAGE on 8% polyacrylamide gels prepared with WIDE RANGE Gel Preparation Buffer (Nacalai Tesque, Shiga, Japan) and stained with CBB G-250. For 2D-PAGE, parts of unstained gels from CN-PAGE were separated by second dimensional electrophoresis. Specifically, each lane was split into a gel strip, and the gel strip was incubated in SDS-sample buffer containing 5% 2-mercaptoethanol for 20 min at 60 °C. The gel strips were then subjected to reducing SDS-PAGE followed by staining with CBB G-250 [40 (link)]. The band intensities were analyzed by ImageJ (http://rsb.info.nih.gov/ij/index.html ; accessed on 1 April 2021). Statistical analyses were performed using Student’s t-test.
5
Gel Filtration Chromatography for Protein Complex Analysis
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Gel filtration chromatography was performed using the SMART system (Amersham Pharmacia). In the experiments testing the PolD–GINS and PolD–GINS–GAN complex formation, each protein and mixture were incubated for 3 min at 60°C. Aliquots (20 μl) of the protein solution was applied on a Superose 6 PC 3.2/30 column (GE Healthcare) and eluted with a buffer containing 50 mM Tris–HCl, pH 8.0 and 0.15 M NaCl. Aliquots (0.1 μl) of the applied solution and the aliquots (4 μl) of each fraction from the eluates were subjected to 10% SDS-PAGE containing WIDE RANGE Gel Preparation Buffer (Nacalai Tesque), followed by silver staining. In these experiments testing the DP1N–Gins51C interaction, each protein (120 μM) and mixture were incubated for 5 min at 60°C, and were applied on a Superdex 200 PC 3.2/30 column (GE Healthcare). The aliquots (4 μl) of each fractionated solution eluted with a buffer containing 50 mM Tris–HCl, pH 8.0 and 0.3 M NaCl were applied Tricine–SDS–10%T, 2.6%C PAGE, followed by CBB staining. The standard marker proteins, including thyroglobulin (670 000), γ-globulin (158 000), ovalbumin (44 000) and myoglobin (17 000), were also subjected to the same gel filtration as controls.
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