Western blot was performed as previously described [25 (link)]. The heart tissues were homogenized on ice for 30 min in a RIPA buffer (Sigma-Aldrich) supplemented with protease inhibitor. The lysates were heated at 95°C for 5 min and loaded on 10% gels (Bio-Rad, San Diego, CA, USA) for sodium dodecyl sulfate–polyacrylamide gel electrophoresis. After electrophoretic separation, the proteins were transferred onto 0.2 µm nitrocellulose membranes (Amersham, Germany), blocked with 5% nonfat milk (in Tris-buffered saline [TBS] + 0.01% Tween) and incubated overnight at 4°C with the following primary antibodies: anti-CD11b (1:1,000; Abcam) and anti-GAPDH (1:1,000; Abcam). The horseradish peroxidase (HRP)-linked secondary antibodies (1:2,000; Abcam) and ECL kit (WBLUR0100; Millipore Corporation, Billerica, MA, USA) were further used to visualize the blots in the membrane. Proteins were finally visualized by an LAS-4000 mini system (Fujifilm, Japan). The intensity of protein bands was quantified with Quantity one software. The proteins of tissue expression were standardized to GAPDH levels.
0.2 m nitrocellulose membrane
Manufactured by Cytiva
Sourced in United States
The 0.2 μm nitrocellulose membranes are a type of laboratory filtration equipment used for the separation and isolation of small particles, molecules, or cells from liquid samples. These membranes have a pore size of 0.2 micrometers, which allows for the effective retention of a wide range of biomolecules while permitting the passage of smaller components.
Automatically generated - may contain errors
Lab products found in correlation
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Ecl kit, by Merck Group (1 mentions)
Ripa buffer, by Merck Group (1 mentions)
Anti cd11b, by Abcam (1 mentions)
Anti gapdh, by Abcam (1 mentions)
Las 4000 mini system, by Fujifilm (1 mentions)
Enhanced chemiluminescence, by Merck Group (1 mentions)
Anti β actin antibody, by Santa Cruz Biotechnology (1 mentions)
Gel pro analyzer version 3, by Media Cybernetics (1 mentions)
Anti tlr2 antibody, by Abcam (1 mentions)
Anti renin, by Santa Cruz Biotechnology (1 mentions)
Bolt 4 12 bis tris plus gel, by Thermo Fisher Scientific (1 mentions)
Multigel 21 imaging system, by Top-Bio (1 mentions)
Odyssey clx, by LI COR (1 mentions)
Complete protease inhibitor cocktail, by Roche (1 mentions)
Amersham imager 680 system, by GE Healthcare (1 mentions)
Horseradish peroxidase hrp conjugated goat anti rabbit igg, by Beyotime (1 mentions)
Anti bcl 2, by Cell Signaling Technology (1 mentions)
Beyoecl star, by Beyotime (1 mentions)
β actin, by Cell Signaling Technology (1 mentions)
5 protocols using 0.2 m nitrocellulose membrane
1
Western Blot Analysis of CD11b in Heart Tissue
2
Quantifying Kidney Protein Expression
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Western blot analysis was performed to measure TLR2, renin, and AT1R protein expression. Kidney sections were homogenized in PRO-PREP protein extraction solution (iNtRON, Seongnam, Korea). Total protein (40 µg) was loaded. Samples were wet transferred onto 0.2 µm nitrocellulose membranes (Amersham Pharmacia, Piscataway, NJ, USA). Blots were blocked for 1 hour with 5% nonfat dry milk in Tris-buffered saline-Tween buffer compose of 20 mM Tris-HCl (pH 7.6), 0.8% NaCl and 0.05% Tween 20, and incubated overnight at 4°C with anti-TLR2 antibody (Abcam, Cambridge, MA, USA), AT1R (Santa Cruz Biotechnology), anti-renin (Santa Cruz Biotechnology) and anti-β-actin antibody (Santa Cruz Biotechnology). Blots were incubated with horseradish peroxidase-conjugated secondary anti-rabbit immunoglobulin G antibody (Cell Signaling Technology) for 1 hour. Bands were detected by enhanced chemiluminescence (Millipore, Billerica, MA, USA) and exposed to film. The optical density for quantification was determined using Gel-Pro Analyzer version 3.1 (Media Cybernetics, Bethesda, MD, USA).
3
Western Blot Analysis of Extracellular Vesicles
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Igepal CA-630, 0.5% sodium deoxycholate, 0.1% SDS) with cOmplete protease inhibitor cocktail (Roche). Protein concentrations of EPs and cell lysates were determined using Pierce™ BCA Protein Assay Kit (Thermo Scientific). Protein lysates were separated using Bolt™ 4-12% Bis-Tris Plus Gels (Invitrogen) and transferred onto 0.2 µm nitrocellulose membranes (Amersham Bioscience, Little Chalfont, UK). The membranes were blocked with 5% BSA (Cyrusbioscience, Taiwan) in PBST (1X PBS, pH 7.4, with 0.1% Tween-20) for 1 h at room temperature and immunoprobed with primary antibodies overnight at 4 °C. The membranes were washed three times in PBST at room temperature, incubated with secondary antibodies for 1 h at room temperature followed by another three washes in PBST. For Western blots of purified sEV pellets from sucrose gradients, the membranes probed by HRP-conjugated secondary antibodies were developed by chemiluminescence using Trident Femto Western HRP Substrate kit (GeneTex) with MultiGel-21
Imaging System (Topbio, Taiwan). For western blots of protein knockdown, EV-293T-GRET/-PalmGRET subtypes, and EP-HCA1-WT/GRET/PalmGRET characterization experiments, the targeted proteins immunoprobed by IR-dye-conjugated secondary antibodies were imaged by ODYSSEY CLx (LI-COR Biosciences, Nebraska, USA). Antibody dilutions and hosts are provided in Table S2.
Imaging System (Topbio, Taiwan). For western blots of protein knockdown, EV-293T-GRET/-PalmGRET subtypes, and EP-HCA1-WT/GRET/PalmGRET characterization experiments, the targeted proteins immunoprobed by IR-dye-conjugated secondary antibodies were imaged by ODYSSEY CLx (LI-COR Biosciences, Nebraska, USA). Antibody dilutions and hosts are provided in Table S2.
4
Western Blot Analysis of Protein Samples
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Protein samples were prepared in lysis buffer (HEPES 25 mmol/L, KAc 150 mmol/L, EDTA pH 8.0 2 mmol/L, NP-40 0.1%, NaF 10 mmol/L, phenylmethylsulfonyl fluoride (PMSF) 50 mmol/L, aprotinin 1 µg/µl, pepstatin 1 µg/µl, leupeptin 1 µg/µl, and DTT 1 mmol/L). Protein concentration was quantified by bicinchoninic acid (BCA) protein assay (Beyotime, Shanghai, China) according to the protocol using bovine serum albumin (BSA) to prepare a standard curve. Gel electrophoresis was performed with 10–20 µg of protein using 4%–15% gels (Beyotime, China), followed by transblotting to 0.2-µm nitrocellulose membrane (Amersham, Piscataway, NJ, USA). Protein band intensities were determined and detected with BeyoECL Star (Beyotime, China) using the Amersham Imager 680 system (GE). Primary antibodies used in the experiments, including anti-Bcl-2 (rabbit mAb, Cell Signaling, Danvers, MA, USA) and β-actin (rabbit mAb, Cell Signaling, USA) were diluted 1:1,000 in 1% BSA. The secondary antibody used in the experiments including horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (Beyotime, China) was diluted at 1:1,000.
5
Slot Blot Quantification of TOP2β
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Samples were diluted fourfold (500 μL total volume) in NaPO4 buffer (25 mM, pH 6.5), and slot blotted onto 0.2 µM nitrocellulose membrane (Amersham), using the Minifold I (Whatman) manifold. The wells were washed twice with 750 µL NaPO4 buffer. The membrane was then blocked with 10% milk-TBST for 1 h at room temperature, prior to incubation overnight with 1/1000 anti-TOP2β antibody (Clone 40, BD Biosciences) at 4 °C. The membrane was washed four times with TBST, incubated with a 1/10000 dilution of HRP-conjugated Rabbit anti-Mouse IgG (ThermoFisher) for 1 h at room temperature, washed four times with TBST, and incubated with ECL detection reagent for 1 min. X-Ray film was used for detection.
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