EV and hybrid EV samples solubilized with sodium dodecyl sulfate (SDS) buffer were separated using 12.5% polyacrylamide gel and transferred to polyvinylidene difluoride (PVDF) membranes. After blocking with PVDF Blocking Reagent for Can Get Signal (TOYOBO Co., Ltd., Osaka, Japan), the membranes were reacted with primary antibodies to PD-1 (ab89828, Abcam, Cambridge, UK) or gp64 (sc-65499, Santa Cruz Biotechnology, CA, USA). After washing with Tris-buffered saline with 0.1% Tween 20 (TBST), the membranes were reacted with horseradish peroxidase-conjugated goat anti-mouse IgG (Santa Cruz Biotechnology). After washing with TBST again, the membranes were reacted with ECL Western blotting detection reagents (GE Healthcare, Chicago, IL, USA) and the band signals were visualized using a LAS-4000 (GE Healthcare).
Pvdf blocking reagent for can get signal
Manufactured by Toyobo
Sourced in Japan
PVDF Blocking Reagent for Can Get Signal is a laboratory product designed to enhance the performance of Western blot analysis. It serves as a blocking agent, effectively reducing non-specific binding of antibodies to the PVDF membrane, thereby improving the signal-to-noise ratio in Western blot experiments.
Automatically generated - may contain errors
Lab products found in correlation
Can get signal solution 1, by Toyobo (6 mentions)
Pvdf membrane, by Bio-Rad (5 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (4 mentions)
Ripa buffer, by Thermo Fisher Scientific (4 mentions)
Ecl western blotting detection reagent, by GE Healthcare (3 mentions)
Las4000, by GE Healthcare (3 mentions)
Protease inhibitor cocktail, by Roche (3 mentions)
Can get signal solution 2, by Toyobo (3 mentions)
Horseradish peroxidase conjugated goat anti mouse igg, by Santa Cruz Biotechnology (2 mentions)
Protease inhibitor cocktail, by Merck Group (2 mentions)
Anti β actin, by Merck Group (2 mentions)
Fusion fx7, by Vilber (2 mentions)
Dc protein assay kit, by Bio-Rad (2 mentions)
Can get signal immunoreaction enhancer solution 1, by Toyobo (2 mentions)
Can get signal immunoreaction enhancer solution 2, by Toyobo (2 mentions)
Lumina forte western hrp substrate, by Merck Group (2 mentions)
Tris buffered saline with tween 20 tbst 10x, by Cell Signaling Technology (2 mentions)
Anti β actin, by Cell Signaling Technology (2 mentions)
Cell counting kit 8 (cck8), by Dojindo Laboratories (2 mentions)
Triton x 100, by Merck Group (2 mentions)
31 protocols using pvdf blocking reagent for can get signal
1
PD-1 and gp64 Protein Detection in EV and Hybrid EV Samples
2
Western Blot Analysis of EphA5 in hBMSCs
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Proteins were extracted from hBMSCs. Total cellular protein was prepared by lysing cells in RIPA buffer at various time points. The protein concentration was determined using the BCA Protein Reagent Kit (Pierce, Rockford, IL). A primary antibody for EphA5 (AP7610d, ABGENT) and α-tubulin (11H10) rabbit mAb (#2125S, Cell Signaling Technology, Inc., Tokyo, Japan) were obtained. Protein (15 μg) was separated by 10% SDS-PAGE and then transferred to a polyvinylidene difluoride (PVDF) membrane. After blocking with PVDF Blocking Reagent for Can Get Signal (TOYOBO Life Science, Tokyo, Japan), membranes were hybridized with the primary antibody overnight at 4°C and then hybridized with HRP-linked anti-rabbit immunoglobulin G secondary antibody (#7074, Cell Signaling Technology, Inc., Tokyo, Japan) for 1 h at room temperature. We then added Lumigen TMA-6 (Lumigen Inc., Southfield, MI, USA) onto the membrane and let it stand for 2 minutes. The signals were detected using an enhanced chemiluminescence method on an ImageQuant LAS4000 Series system (GE Healthcare UK Ltd., England).
3
Western Blot Analysis of SMPD1
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The reagents used were as follows: RIPA buffer for preparing cell lysates (Thermo Fisher Scientific Inc., Waltham, MA, USA), Protease Inhibitor Cocktail (Sigma-Aldrich Co. LLC, St. Louis, MO, USA), polyacrylamide gels for SDS-PAGE (Wako Pure Chemical Industries, Ltd. Osaka, Japan), PVDF membrane (Bio-Rad Laboratories, Inc., Hercules, CA, USA), PVDF Blocking Reagent for Can Get Signal (TOYOBO CO., LTD., OSAKA, JAPAN), and Luminata Forte Western HRP Substrate (Millipore Corporation, Billerica, MA, USA). The immunoblots were visualized by Fusion-FX7 (Vilber Lourmat, Marne-la-Vallée, France). Primary antibodies used were as follow anti-SMPD1 (Proteintech Group, Inc., Chicago, USA, 14609–1-AP) and anti-β-actin (Sigma-Aldrich Co. LLC, A2228). Also, β-actin was used as an internal control.
4
Western Blot Analysis of Protein Samples
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Cell and tissue lysates were prepared with 1% SDS buffer. The protein concentrations were measured by using a DC Protein Assay Kit (Bio-Rad). Protein samples (1–5 μg/lane) were resolved in 10%–12.5% SDS-PAGE gells and transferred to 0.45 μm PVDF membranes (Immobilon-P Membrane, EMD Millipore). After having been blocked with PVDF Blocking Reagent for Can Get Signal (TOYOBO) for 1 hour, the membranes were incubated overnight at 4°C with primary antibodies. The primary antibodies used for immunoblotting are summarized in Supplemental Table 2 . The membranes were incubated at room temperature with HRP-linked anti–mouse IgG or anti–rabbit IgG as secondary antibodies (Cell Signaling Technology) diluted 1:4000. After 3 washes with TBS-T, the immunoblots were visualized by using the Luminata Forte Western HRP substrate (EMD Millipore). The loaded amount was verified with an anti–β-actin mouse monoclonal antibody (clone AC-74, Sigma-Aldrich, A5316). Please see Supplemental Methods for detailed information.
5
Western Blotting of Cell Lysates
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Neurons were lysed in RIPA Buffer with cOmplete, Mini, EDTA-Free Protease Inhibitor Cocktail (Roche) and PhosSTOP Phosphatase Inhibitor Cocktail (Roche) on ice for 1 hr with regular vortexing to aid lysis. Insoluble proteins were removed via centrifugation, and lysate protein concentration was determined using the Pierce Bicinchoninic Acid Protein Assay Kit (Invitrogen) using a standard curve created with BSA standards of known concentration. Equal quantities of protein (20–50 µg) were resolved on 4–20% gradient SDS-Polyacrylamide gels (Bio-Rad) and then transferred onto Polyvinylidene difluoride membranes (Millipore Sigma). Membranes were blocked in PVDF Blocking Reagent for Can Get Signal (Toyobo) for 1 hr. Primary antibodies were diluted in Can Get Signal Immunoreaction Enhancer Solution 1 (Toyobo) and membranes were incubated overnight at 4°C. HRP-labeled secondary antibodies were diluted in Can Get Signal Immunoreaction Enhancer Solution 2 (Toyobo) and membranes were incubated for 1 hr at room temperature. Blots were developed using Western Lightning Plus-ECL Enhanced Chemiluminescence Substrate (PerkinElmer) and ProSignal ECL Blotting Film (Prometheus Protein Biology Products) according to manufacturer’s instructions. Blots were stripped for reblotting using NewBlot PVDF Stripping Buffer (Licor). Band density was quantified in ImageJ.
6
Western Blot Analysis of HCP-1 Protein
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Hematoporhyrin dihydrochloride (Wako Pure Chem. Ind., Ltd., Osaka, Japan), Cell Counting Kit-8 (DOJINDO LABORATORIES, Kumamoto, Japan), Trizma® base (Sigma-Aldrich Japan K.K., Tokyo, Japan), NaCl (Wako Pure Chem. Ind., Ltd., Osaka, Japan), Triton X-100 (Sigma-Aldrich Japan K.K.) sodium dodecyl sulfate (SDS) (Wako Pure Chem. Ind., Ltd.), deoxycholic acid (Wako Pure Chem. Ind., Ltd.), hydrochloric acid (Wako Pure Chem. Ind., Ltd.), NuPAGE® Novex® 12% Bis-Tris gels (Life Technologies Japan Ltd., Tokyo, Japan), PVDF Blocking Reagent for Can Get Signal® (TOYOBO CO., LTD., Osaka, Japan), Tris Buffered Saline with Tween® 20 (TBST-10X, Cell Signaling Technology Japan, K.K., Tokyo, Japan), HCP-1 antibody (Santa Cruz Biotechnology, Inc., Dallas, TX), Can Get Signal® Immunoreaction Enhancer Solution 1 (TOYOBO CO., LTD.) horseradish peroxidase (HRP) linked anti-rabbit IgG antibody (Cell Signaling Technology Japan, K.K.), Lumina forte western HRP substrate (Millipore Co., Billerica, MA), anti-β-actin (Cell Signaling Technology Japan, K.K.), and Can Get Signal® Immunoreaction Enhancer Solution 2 (TOYOBO CO., LTD.) were purchased and used without further purification or modification.
7
Detecting Influenza Virus Proteins
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Virus-infected AX4/PB2 cells were lysed by RIPA buffer with protease inhibitors, and the cell lysates were mixed with 4× sample buffer (Invitrogen) and 100 mM DTT. Samples were subjected to SDS/PAGE, and proteins were electrophoretically transferred to a polyvinylidene difluoride membrane (Millipore). The membrane was blocked with PVDF Blocking Reagent for Can Get Signal (Toyobo) and was then incubated with anti-influenza virus (R309) rabbit polyclonal antibodies, which were prepared in our laboratory, followed by six washes with TBS with Tween 20 (TBS-T). Finally, the membrane was incubated with a secondary antibody, AP-conjugated anti-rabbit IgG (Cell Signaling Technology), and was washed six times with TBS-T. Specific proteins were detected using 1-Step NBT/BCIP solution (Thermo). β-Tubulin as an internal control was detected with anti-β-Tubulin (Cell Signaling Technology).
8
Western Blot Analysis of Cellular Proteins
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Western blot analysis was performed, as described previously68 (link). Cells were washed thrice with phosphate-buffered saline and lysed in radioimmunoprecipitation assay (RIPA) buffer (Nacalai Tesque). Cell lysates (10–20 μg) were heated in sodium dodecyl sulfate (SDS) sample buffer (125 mM Tris–HCl, pH 6.8, 4% SDS, 25% glycerol, 10% 2-mercaptoethanol, 0.05 mM phenylmethanesulfonyl fluoride, and 0.004% bromophenol blue), separated by 10% or 15% e-PAGEL (Atto Corp, Tokyo, Japan) according to the manufacturer’s recommendations, and transferred onto polyvinylidene fluoride (PVDF) immuno-blot membranes (Bio-Rad). The membranes were blocked with PVDF Blocking Reagent for Can Get Signal (Toyobo) for 1 h at 20–25 °C and incubated with the appropriate primary antibody in Can Get Signal Solution 1 (Toyobo) overnight at 4 °C. After washing, the membranes were incubated with the appropriate secondary antibody for 1 h at 20–25 °C. The signal was visualized by Chemi-Lumi One (Nacalai Tesque) and analyzed by ChemiDoc XRS + system with Image Lab software (Bio-Rad).
9
Exosome Identification and Characterization
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Intact EVs were isolated and concentrated as described in the procedure for NTA. They were mixed with 4x NuPAGE LDS sample buffer and 10x Sample Reducing Agent (Thermo Fisher Scientific, MA, USA). The mixture was incubated at 70 °C for 10 min for reduction of disulfide bond. Proteins were separated by electrophoresis with NuPAGE SDS-PAGE system (Thermo Fisher Scientific) with protein size standard (Bio-Rad Laboratories, CA, USA) and blotted to PVDF membrane (Bio-Rad Laboratories). The blot was blocked with PVDF Blocking Reagent for Can Get Signal (TOYOBO, Osaka, Japan). To identify isolated EVs as exosomes, primary antibodies against exosome-enriched protein markers CD9, CD81 (mouse mAb from Novus Biologicals, CO, USA), and Flotillin-1 (mouse mAb from BD Biosciences, NJ, USA) were used. All of them were diluted 1000-fold in Can Get Signal solution 1. HRP-conjugated anti-mouse secondary antibodies (Cell Signaling Technology, MA, USA) were used with dilution of 5000-fold in Can Get Signal solution 2.
10
Western Blot Analysis of GDF15 Protein
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Cell lysates were separated by SDS-PAGE using a 10–20% gradient gel (DRC, Tokyo, Japan). Proteins were transferred to 0.2-μm PVDF membranes (Millipore, MA, USA) at 10V overnight. The membranes were blocked with PVDF Blocking Reagent for Can Get Signal (TOYOBO, Osaka, Japan) for 1 h. Anti-GDF15 rabbit polyclonal antibody (L300; Cell Signaling Technology, MA, USA) diluted 1:1000 and anti-β-actin goat polyclonal antibody (C-11; Santa Cruz Biotechnology, TX, USA) diluted 1:500 were used as primary antibodies. Horseradish peroxidase (HRP)-linked antirabbit IgG donkey antibody (GE Healthcare, Buckinghamshire, UK) diluted 1:2000 and HRP-linked antigoat IgG rabbit antibody (MP Biomedicals, OH, USA) diluted 1:2000 were used as a secondary antibody. These primary and secondary antibodies were diluted with Can Get Signal immunoreactions enhancer solution (TOYOBO). Antigens on the membrane were detected with chemiluminescence detection reagents (Michigan Diagnostics, MI, USA).
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