Lysate of tooth germ at E18 (30 µg/lane) was separated under reducing conditions on 5% SDS polyacrylamide gel (Super Sep, Wako Pure Chemical Industries, Osaka, Japan) and transferred to PVDF membrane (Atto Corp., Tokyo, Japan). The blot was blocked with 5% BSA in Tris-buffered saline with 0.2% tween 20 (TBST) overnight at 4°C and incubated with anti-Tie2 or anti-phosphorylated Tie2 antibodies (1:1000, Merck Millipore, Darmstadt, Germany) for 3 hr at RT. After washing with TBST, HRP-conjugated anti-rabbit or anti-mouse antibodies (1:2000, Amersham Biosciences Corp., Piscataway, NJ, USA) were applied and incubated for 1 hr at RT. Following the final wash, the blot was incubated with ECL Prime Western Blotting Detection Reagent and the signal was detected with image analyzer, ImageQuant LAS 4000mini (both from GE Healthcare UK Ltd., Buckinghamshire, UK).
Supersep
Manufactured by Fujifilm
Sourced in Japan
SuperSep is a laboratory equipment product manufactured by Fujifilm. It is designed for the separation and purification of biological samples. The core function of SuperSep is to facilitate the efficient separation of target molecules or cells from complex mixtures, such as cell lysates or biological fluids.
Automatically generated - may contain errors
Lab products found in correlation
P egfr, by Cell Signaling Technology (2 mentions)
Polyvinylidene fluoride membrane, by Merck Group (2 mentions)
Polyvinylidene difluoride membrane, by Merck Group (2 mentions)
Pvdf membrane, by ATTO Corporation (1 mentions)
Imagequant las 4000 mini, by GE Healthcare (1 mentions)
Amersham imager 600, by GE Healthcare (1 mentions)
Pvdf membrane, by GE Healthcare (1 mentions)
Amersham imager, by Cytiva (1 mentions)
Complete mini, by Roche (1 mentions)
Immobilon p membrane, by Merck Group (1 mentions)
Imagequant las 4000 mini imaging system, by GE Healthcare (1 mentions)
Multi gauge version 3, by GE Healthcare (1 mentions)
Horseradish peroxidase conjugated anti mouse igg antibody, by Promega (1 mentions)
Las 4000, by Cytiva (1 mentions)
P akt, by Cell Signaling Technology (1 mentions)
Protease inhibitor, by Merck Group (1 mentions)
Phosphatase inhibitor, by Merck Group (1 mentions)
Radioimmunoprecipitation assay buffer, by Merck Group (1 mentions)
Pher2, by Abcam (1 mentions)
β actin, by Cell Signaling Technology (1 mentions)
11 protocols using supersep
1
Tie2 Phosphorylation in Tooth Germ Development
2
Western Blot Analysis of Protein Targets
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The proteins (20 µg) were separated using 7.5% polyacrylamide gel electrophoresis (SuperSep, WAKO, Osaka, Japan) and blotted onto a polyvinylidene difluoride (PVDF) membrane (GE Healthcare Life Sciences). The membrane was blocked with 5% skim milk in TBS containing 0.1% Tween-20 (TBS-T) for 1 h at room temperature and then incubated at 4 °C overnight with primary antibody: R-10G anti-KS antibody (1:200 dilution) or the rabbit polyclonal anti-Ptprz-S antibody (1:500 dilution)34 (link) in 5% skim milk/TBS-T overnight at 4 °C. Membranes were washed with TBS-T and incubated for 30 min at room temperature with HRP-conjugated secondary antibodies. Bound antibodies were detected with a Novex ECL Chemiluminescent reagent kit (Thermo Fisher Scientific) and an Amersham Imager 600 (GE Healthcare Life Sciences). Densitometry analysis of immunoreactive bands was performed with the ImageJ software (http://imagej.nih.gov/ij/ ) (National Institutes of Health, Bethesda, MD).
3
Feline Coronavirus Nucleocapsid Protein Detection
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The cell membranes were disrupted with cell lysis buffer (10 mM Tris-HCl, pH 7.8, 1 mM ethylenediaminetetraacetic acid (EDTA), 1% NP-40, 0.15 M NaCl), including cOmplete Mini (Roche Diagnostics, Tokyo, Japan) at 20 h after infection. The cell lysates were resolved by electrophoresis on 12.5% SuperSep gels (Fujifilm WAKO Pure Chemical Corporation) and Western blotting onto Immobilon-P membranes (Millipore, Tokyo, Japan). Non-specific protein binding was blocked with 5% non-fat dry milk for 1 h, and then the membranes were incubated with anti-feline coronavirus nucleocapsid (N) antibody (FIPV3-70; MyBioSource, San Diego, CA, USA) or anti-glyceraldehyde-3-phosphate dehydrogenase antibody (GAPDH, 6c5 clone; Calbiochem, Tokyo, Japan) for 1 h. Antigen signals were visualized by reacting proteins on the membranes with horseradish peroxidase-conjugated anti-mouse IgG antibody (Promega Corporation, Madison, WI, USA), followed by an enhanced chemiluminescence substrate (ImmunoStar LD; Fujifilm WAKO Pure Chemical Corporation, Tokyo, Japan), according to the manufacturer’s protocol. Signals were detected using the ImageQuant LAS 4000-mini Imaging System (GE Healthcare Life Sciences, Marlborough, MA, USA) and analyzed using Multi Gauge Version 3.0 software (GE Healthcare Life Sciences, MA, USA).
4
Phosphorylation Analysis of Bdp1 Protein
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Proteins from synchronized cells samples, prepared as previously described, were precipitated by using TCA. After washing twice with 100% ethanol, proteins were resuspended in 1 M Tris–HCl pH 8 buffer. Equal amounts of proteins were used to load the gels. After heat denaturation at 95 °C for 5 min, debris was discarded by centrifugation and loaded into the gel. Ten percent acrylamide Phos-tag gel electrophoresis run for 6 h at 150 V (Supersep, Wako chemicals), followed by three washes for 10 min with transfer buffer containing 5 mM EDTA and three washes for 10 min with transfer buffer without containing EDTA. The gel was transfered to a membrane and phosphorylation of Bdp1 was finally monitored by western blotting as described previously (38 (link)) using Myc antibodies.
5
Western Blot Analysis of Signaling Proteins
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Cells were harvested and lysed in radioimmunoprecipitation assay buffer (Sigma-Aldrich) containing phosphatase inhibitors (Sigma-Aldrich) and protease inhibitors (Sigma-Aldrich) for 15 min on ice. Cell lysates (15-20 mg/mL) were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis using Super Sep (Wako, Osaka, Japan) and transferred onto polyvinylidene difluoride membranes, which were blocked for 1 h and incubated with the respective primary antibody overnight at 4°C. The antibody against GPNMB was from R&D Systems (Minneapolis, MN, USA), antibodies against HER2 and phosphorylated HER2 (p-HER2) were purchased from Abcam (Cambridge, UK), and all other antibodies [EGFR, p-EGFR, p42/44 MAPK (ERK), p-p42/44 MAPK (ERK), v-AKT murine thymoma viral oncogene homolog (AKT), p-AKT, and β-actin] were obtained from Cell Signaling Technology (Danvers, MA, USA). The membranes were then washed and incubated with the appropriate secondary antibodies, and immunoreactive proteins were visualized and captured by an LAS-4000 camera system (FUJIFILM, Tokyo, Japan).
6
Nuclear Protein Extraction and Immunoblotting
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One day after treatment with FAD, the hfNSCs were washed with cold PBS and hypotonic buffer (20 mm Tris/HCl pH 7.4, 10 mm NaCl, 1.5 mm MgCl2) added. The nuclear extract was retrieved by centrifugation (800 g ). Pellet samples (nuclei) and supernatant (without nuclei; almost purely cytoplasm) for immunoblotting were prepared as follows. Pellets were lysed with lysis buffer (50 mm Tris/HCl pH 7.4, 150 mm NaCl, 1% Triton X‐100) that included a protease inhibitor cocktail (Nacalai Tesque, Inc.) and a phosphatase inhibitor cocktail (Nacalai Tesque, Inc.) for 30 min on ice. Then, sample buffer (Nacalai Tesque, Inc.) was added to the lysed samples, and the samples were incubated for 5 min at 95 °C. Aliquots of total cell lysate were separated by 5–20% SuperSep (Wako Pure Chemical Industries, Ltd., Osaka, Japan) and transferred onto polyvinylidene fluoride membranes (EMD Millipore). After blocking with BlockingOne (Nacalai Tesque, Inc.) containing 0.1% Tween 20, the membranes were incubated with appropriate primary antibodies. After washing with TBST (Tris‐buffered saline, 0.1% Tween 20), the membranes were then incubated with the appropriate HRP‐conjugated secondary antibodies (Abcam), washed, and developed with ECL Prime reagents (GE Healthcare Bio‐Sciences, Pittsburgh, PA, USA).
7
Hemagglutinin Protein Detection Assay
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Pseudotyped VSVs were treated with or without trypsin (final concentration 0.0005%) for 30 min at 37 °C and then mixed with SDS-PAGE sample buffer with 5% 2-mercaptoethanol and boiled for 5 minutes. After electrophoresis on 5–20% SuperSep (Wako), separated proteins were blotted on a polyvinylidene difluoride membrane (Millipore). The membrane was incubated with an anti-H3N2 chicken polyclonal antiserum or anti-HA2 monoclonal antibody 3N12-6-4, which reacts to H1, H2, H5, H6, H17, and H18 HAs, followed by incubation with peroxidase-conjugated rabbit anti-chicken IgY (H+L) or goat anti-mouse IgG (H+L) (Jackson ImmunoResearch). The bound antibodies were visualized with Immobilon Western (Millipore).
8
Western Blot Analysis of Signaling Proteins
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Treatment of the specimen was as described previously (25 (link)–27 (link)). The cell lysate was boiled in sample buffer solution (Wako). Total cell protein extracts (20 μg/lane) were separated by sodium dodecylsulface-polyacrylamide gel electrophoresis using SuperSep™ (Wako) and were electrophoretically transfected onto polyvinyl difluoride menmbranes. The membranes were blocked with PVDF blocking reagent (Toyobo, Osaka, Japan) for 1 h. The membrane was then incubated with primary antibodies against β-actin, phosphorylated type of extracellular signal-regulated kinase (p-ERK), c-jun N-terminal kinase (p-JNK), p-AKT, mammalian target of rapamycin (p-mTOR), hepatocyte growth factor receptor (p-cMet), epithelial growth factor receptor (p-EGFR) and caspase-3 (1:5,000; Cell Signaling Technology, Danvers, MA, USA) and EGFR inhibitor (1:5,000; Calbiochem 324674, Merck KGaA, Germany) overnight at 4°C. The primary antibodies were diluted with Can Get Signal Solution 1 (Toyobo). The membrane were then washed with Dako washing buffer (Dako, Glostrup, Denmark) and incubated with the appropriate secondary antibodies (1:25,000; Millipore, Darmstadt, Germany), which were diluted with Can Get Signal Solution 2. The immunoreactive proteins were visualized by chemiluminescence using a LAS-4000 (Fuji film, Tokyo, Japan) and analyzed using ImageJ software (NIH, Bethesda, MD, USA).
9
Immunoblotting of NHEK Proteins
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Cultured NHEKs were lysed with radioimmunoprecipitation assay buffer containing protease inhibitors (Roche) for 30 min on ice. Aliquots of the supernatants under reducing conditions were applied to SDS‐5%–20% polyacrylamide gels (SuperSep™; FUJIFILM Wako Pure Chemical) and transferred onto polyvinylidene fluoride membranes (Millipore). The membranes were incubated with optimally diluted antibodies for 1 h at room temperature. After washing, the membranes were reacted with a peroxidase‐labeled secondary antibody for 45 min. Signals were visualized using an enhanced chemiluminescence detection system (Amersham Life Science). The levels of intensity of signals detected in immunoblots were quantified using NIH ImageJ software and standardized to the corresponding levels of β‐actin.
10
Filovirus VLP Production and Western Blot Analysis
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Serum samples were analyzed by western blotting as described previously [14] .
293T cells were transfected with plasmids encoding filovirus (Zaire, Sudan, Tai Forest, Bundibugyo, Reston, and Angola) GP, viral nucleoprotein (NP) and matrix protein (VP40) genes to generate virus-like particles (VLPs). At 48 hours post-transfection, VLPs were recovered from the pellets after centrifugation at 28,000 X g at 4 °C for 1.5 hours through a 25% sucrose cushion. Supernatants from 293T cells transfected with an empty vector, pCAGGS, were used as a negative control. VLPs were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis under non-reducing conditions on 5-20% SuperSep (Wako) and blotted on a polyvinylidene difluoride membrane (Millipore). Bat serum samples diluted at 1:100 were used as primary antibodies, followed by detection with goat anti-bat IgG-heavy and light chain antibody conjugated with horseradish peroxidase (Bethyl). Mouse monoclonal antibodies ZGP42/3.7 to Ebola virus GPs and AGP127-8 to Marburg virus GP were used as positive control antibodies, followed by detection with goat anti-mouse IgG-heavy and light chain antibody conjugated with horseradish peroxidase (Jackson ImmunoResearch) [14] . The bound antibodies were visualized with Western Lightning Plus-ECL (PerkinElmer) and detected by an ImageQuant LAS4000 (GE Healthcare).
293T cells were transfected with plasmids encoding filovirus (Zaire, Sudan, Tai Forest, Bundibugyo, Reston, and Angola) GP, viral nucleoprotein (NP) and matrix protein (VP40) genes to generate virus-like particles (VLPs). At 48 hours post-transfection, VLPs were recovered from the pellets after centrifugation at 28,000 X g at 4 °C for 1.5 hours through a 25% sucrose cushion. Supernatants from 293T cells transfected with an empty vector, pCAGGS, were used as a negative control. VLPs were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis under non-reducing conditions on 5-20% SuperSep (Wako) and blotted on a polyvinylidene difluoride membrane (Millipore). Bat serum samples diluted at 1:100 were used as primary antibodies, followed by detection with goat anti-bat IgG-heavy and light chain antibody conjugated with horseradish peroxidase (Bethyl). Mouse monoclonal antibodies ZGP42/3.7 to Ebola virus GPs and AGP127-8 to Marburg virus GP were used as positive control antibodies, followed by detection with goat anti-mouse IgG-heavy and light chain antibody conjugated with horseradish peroxidase (Jackson ImmunoResearch) [14] . The bound antibodies were visualized with Western Lightning Plus-ECL (PerkinElmer) and detected by an ImageQuant LAS4000 (GE Healthcare).
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