SUN2-containing protein complexes were purified, resolved by 10–20% SDS-PAGE, and stained with a mass spectrometry-compatible Silver Stain MS kit (Wako Pure Chemical Industries Ltd.). Corresponding bands were cut out for trypsinization. Tandem mass spectra of the trypsinized peptides were obtained using a nano LC (UltiMate 3000; Thermo Scientific Dionex, CA, USA) equipped with an L-column 2 C18 (150 mm × 0.075 mm i.d. analytical column, containing 3-µm particles; Chemicals Evaluation and Research Institute, Japan), coupled with a nano-ESI (electrospray ionization)-IT (ion trap)-MS (mass spectrometer) equipped with a nano-ESI ion source (HCTultra; Bruker Daltonik GmbH, Germany). The raw data files were analyzed using Mascot MS/MS Ion Search (Matrix Science, London, UK) and searched against the Swiss-Prot database of human proteins.
Silver stain ms kit
Manufactured by Fujifilm
Sourced in Japan
The Silver Stain MS Kit is a laboratory product designed for the detection and visualization of proteins in polyacrylamide gels. It utilizes a silver-based staining method to enhance the contrast and sensitivity of protein bands, allowing for their identification and analysis. The kit provides the necessary reagents and protocols to perform this staining process.
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Lab products found in correlation
Ubiquitin amc, by R&D Systems (2 mentions)
Lys63 linked ubiquitin tetramers, by R&D Systems (2 mentions)
Prism 9, by GraphPad (2 mentions)
Varioskan lux, by Thermo Fisher Scientific (2 mentions)
Cfi plan fluor series, by Nikon (2 mentions)
Eclipse e600, by Nikon (2 mentions)
D5500, by Nikon (2 mentions)
Ecl select western blotting detection kit, by GE Healthcare (2 mentions)
Complete edta free protease inhibitor cocktail, by Roche (2 mentions)
Anti dykddddk flag tag monoclonal antibodies, by Fujifilm (2 mentions)
Mascot ms ms ion search, by Matrix Science (1 mentions)
Hct ultra, by Bruker (1 mentions)
Peptide calibration standard, by Bruker (1 mentions)
Sequencing grade modified trypsin, by Promega (1 mentions)
Axima cfr plus, by Shimadzu (1 mentions)
Fluoview fv10i, by Olympus (1 mentions)
Anti flag, by Merck Group (1 mentions)
Polyethylenimine (pei), by Polysciences (1 mentions)
Lipofectamine 2000 reagent, by Thermo Fisher Scientific (1 mentions)
Anti usp7, by Fortis Life Sciences (1 mentions)
41 protocols using silver stain ms kit
1
Purification and Mass Spectrometry of SUN2 Complexes
2
Immunoprecipitation of HA-Tagged Proteins
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Organelle fractions from ETMP30-HA and mock pEhEx-HA control homogenates were prepared and approximately 2 μg of proteins were solubilized in 2% digitonin in IP Buffer containing 50 mM BisTris-HCl, pH 7.2, 50 mM NaCl, 0.001 % Ponceau S, and 10 % w/v glycerol for 30 min at 4 °C. The solubilized fraction was collected by centrifugation at 20,000× g for 30 min at 4 °C. Immunoprecipitation was performed as previously described [5 (link)]. Bound proteins were eluted overnight and loaded on SDS-PAGE gels, followed by immunoblotting using mouse anti-HA antibody. Silver staining was performed using Silver stain MS kit (Fujifilm Wako Pure Chemical Corporation, Osaka, Japan), according to the manufacturer’s protocol.
3
Silver Staining for Mass Spectrometry
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For silver staining, plasma (10 μL) was subjected to IEF and then SDS–PAGE without CyDye labeling. The gel was stained using a Silver Stain MS kit (Wako, Tokyo, Japan) in accordance with the manufacturer’s instructions. The gel pieces were excised, destained, washed twice with deionized water and four times with 50 mM ammonium bicarbonate: acetonitrile (1:1), and dehydrated once with acetonitrile. Then, the gel pieces were twice alternately rehydrated with 100 mM ammonium bicarbonate and dehydrated with acetonitrile, and dried by vacuum centrifugation. Protein samples were digested at 37 °C for 12 h with 5 μL of 0.02 μg/μL Sequencing Grade Modified Trypsin (Promega) dissolved in 25 mM ammonium bicarbonate. Peptides were extracted from the gels in 40 μL of 1% trifluoroacetic acid/50% acetonitrile solution by sonication. Samples were spotted onto a μFocus MALDI plate (900 μm, 384 circles, Hudson Surface Technology; Old Tappan, NJ, USA) with an equal volume of matrix solution, containing 10 mM α-cyano-4-hydroxycinnamic acid in 1% trifluoroacetic acid/50% acetonitrile. Positive ion mass spectra were obtained using an AXIMA-CFR Plus (Shimadzu, Kyoto, Japan) in a reflectron mode. MS spectra were acquired over a mass range of 700–4000 m/z and calibrated using peptide calibration standards (∼1,000–3,200 Da, Bruker Daltonics, Yokohama, Japan) [12] (link).
4
MALDI-TOF Protein Identification Protocol
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For protein identification using MALDI‐TOFMS, each tissue homogenate sample (100–150 μg) was subjected to 2D PAGE (mentioned above) without CyDye labeling. To get better resolutions, some samples were separated on larger 2D systems using longer strips (24 cm, pH 3–10 NL [nonlinear]) and larger PAGE gels (257 × 200 × 1 mm; Perfect NT Gel W from DRC). After electrophoresis, the gel was stained using a Silver Stain MS Kit (Wako).
5
Immunoprecipitation and Mass Spectrometry Analysis
6
Protein Identification via Silver Staining
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Eluted samples were electrophoresed and analyzed by silver staining using the Silver Stain MS kit (Wako, Japan) according to the manufacturer's directions. The stained 50-KDa bands were analyzed by Nano LC MS/MS. This analysis was conducted at Japan Bio Services Co., LTD. (Saitama, Japan).
7
Proteomic Analysis of FUT8-KO HepG2 Cells
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Immunoprecipitated samples were separated by polyacrylamide gel electrophoresis and stained with a Silver Stain MS Kit (FUJIFILM Wako Pure Chemical Corporation) in accordance with the manufacturer’s instructions. Mass spectrometry analysis of silver-stained bands of interest that varied between WT and FUT8-KO HepG2 cells was performed by the Joint Research Center for Medical Research and Education at Osaka University, Osaka, Japan. To confirm our mass spectrometry data, WT and VIP36-KO HepG2 cells were treated with mouse anti-MRP2 and rabbit anti-AFP antibodies to analyze the localization of AFP in the bile duct-like structures by confocal microscopy (FLUOVIEW FV10i, OLYMPUS). Visualization with fluorescent labeling was performed using secondary antibodies as described in the immunohistochemistry section.
8
Generation and Characterization of CRY1/CRY2 Antibodies
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Anti-CRY1 or anti-CRY2 polyclonal antibody was raised in rabbits by using partial fragments of mouse CRY1 (506–606) or mouse CRY2 (524–592) as antigen peptides, respectively [15 (link)]. Other antibodies were obtained from the following commercial vendors: anti-USP7 (Bethyl Laboratories), anti-Myc, anti-ubiquitin, anti-GFP (Santa Cruz Biotechnology) and anti-FLAG (Sigma Aldrich). Transfection into the cultured cells was performed with Lipofectamine 2000 reagent (Life Technologies) or polyethylenimine (Polysciences) with standard protocols. USP7 specific inhibitor HBX 41108 was purchased from Boston Biochem and solved in DMSO, which was used as a vehicle control of the inhibitor treatment. Silver stain MS kit (WAKO) was used for silver staining of gels according to the manual.
9
SDS-PAGE Protein Separation and Identification
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Protein samples were separated by SDS–polyacrylamide gel electrophoresis (SDS-PAGE) using 10, 12.5, 15, or 4 to 12% gradient polyacrylamide gel (ATTO) under reducing conditions. A one-fourth volume of 5× SDS-sample buffer [250 mM tris-HCl, 10% SDS, 100 mM DTT, 50% glycerol, and a trace of bromophenol blue at (pH 6.8)] was added to the protein sample and heated at 70°C for 10 min. For the MS analysis, 1 μl of 1% acrylamide monomer was added to the samples after heating to form a propionamide group on the reduced free sulfide of Cys residues. After electrophoresis, protein bands were visualized by Coomassie brilliant blue staining or a Silver Stain MS kit (Wako Pure Chemical, Osaka, Japan). For mass spectrometric sequencing, the excised band from the gel was cut and destained, followed by drying.
10
Cell Lysis and Immunoblotting Protocols
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For two-dimensional culture, cells were lysed in n-octyl-β-D-glucopyranoside (ODG) buffer (20 mM Tris–HCl [pH 7.4], 150 mM NaCl, 1 mM EDTA, 1 mM Na3VO4, 20 mM NaF, 1% Nonidet P-40, 5% glycerol, 2% ODG and a protease inhibitor cocktail [Nacalai Tesque]), and immunoblotting was performed. For three-dimensional culture, the cyst-containing collagen matrix was incubated with HBS buffer (10 mM Hepes [pH 7.3], 140 mM NaCl, 4 mM KCl, 1.8 mM CaCl2, and 1 mM MgCl2) containing 0.1% collagenase (Roche) at 37°C. Cysts were harvested by centrifugation and lysed in SDS sample buffer (50 mM Tris–HCl [pH 6.8], 2% SDS, 100 mM NaCl, 1 mM EDTA, 1 mM Na3VO4, 20 mM NaF and 5% sucrose) before immunoblotting. For immunoprecipitation assays, cells were lysed in ODG buffer and the lysates were incubated with a specific antibody for 1 h at 4°C. Immunoprecipitated proteins were pulled down with protein A- or G-sepharose (GE Healthcare) for immunoblotting. HRP-conjugated anti-mouse or anti-rabbit IgG (Zymed) was used as the secondary antibody. All immunoblots were visualized and quantitated using a Luminograph II System (Atto). Silver staining was performed using the Silver Stain MS Kit (Wako).
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