Treated and untreated cortical neurons or neuronally differentiated PC12 cells were lysed and proteins were analyzed by western blotting [50 (link)]. 30-50 μg of protein for each condition was resolved in 8–12% SDS-PAGE as per the need followed by their transfer on to PVDF membrane (Hybond: GE Healthcare, Buckinghamshire, UK). The proteins were then probed with the desired primary antibodies at 4 °C for overnight. HRP-conjugated secondary antibodies against the primary antibodies were used. The blots were detected using Biorad western blotting detection reagent, according to the manufacturer’s protocol. Imaging of all Western blots was performed using an Azure Chemidoc system using manufacturer’s protocol.
Western blotting detection reagent
Manufactured by Bio-Rad
Sourced in United States
Western blotting detection reagents are laboratory products used to visualize and quantify specific proteins in a sample. They enable the detection and analysis of target proteins on a western blot membrane.
Automatically generated - may contain errors
Lab products found in correlation
Nitrocellulose membrane, by Bio-Rad (2 mentions)
Ab51037, by Abcam (1 mentions)
Ab103590, by Abcam (1 mentions)
Ab16663, by Abcam (1 mentions)
Ab32053, by Abcam (1 mentions)
β actin, by Thermo Fisher Scientific (1 mentions)
Hybond p polyvinylidene difluoride membrane, by Cytiva (1 mentions)
Anti cleaved caspase 3 9661, by Cell Signaling Technology (1 mentions)
Sc 9081, by Santa Cruz Biotechnology (1 mentions)
Enhanced chemi luminescence (ecl), by Bio-Rad (1 mentions)
Kojic acid, by Merck Group (1 mentions)
Resazurin, by Merck Group (1 mentions)
3 4 dihydroxy l phenylalanine, by Merck Group (1 mentions)
Phosphatase inhibitor cocktail, by Merck Group (1 mentions)
Quantipro bca assay kit, by Merck Group (1 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
Mushroom tyrosinase, by Merck Group (1 mentions)
Igf 1, by Cell Signaling Technology (1 mentions)
P jak2, by Cell Signaling Technology (1 mentions)
P stat5, by Cell Signaling Technology (1 mentions)
10 protocols using western blotting detection reagent
1
Western Blotting Analysis of Neuronal Proteins
2
Western Blot Analysis of Protein Expression
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Cells were harvested and lysed in a RIPA buffer, and centrifuged at 16,000× g for 20 min at 4 °C. The supernatants were collected to determine protein concentration using the Bradford method. Equal amounts of protein were subjected to 8–10% SDS-PAGE for 2 h at 100 V. The separated proteins were transferred onto Hybond-P+ polyvinylidene difluoride membranes (Amersham, Buckinghamshire, UK). The membranes were blocked with 5% nonfat milk in Tris-buffered saline containing 0.05% Tween-20 (TBS-T) for 1 h at room temperature, and then incubated with the following primary antibodies: anti-cyclin D1 (ab16663; 1:1000, Abcam), anti-cyclin B1 (ab32053; 1:1000, Abcam), anti-cleaved caspase-3 (9661S; 1:1000, Cell Signaling Technology), anti-pH2AX (2577S; 1:1000, Cell Signaling Technology), anti-CD44 (ab51037; 1:1000, Abcam), anti-ESM1 (ab103590; 1:1000, Abcam), and anti-OCT3/4 (sc-9081; 1:1000, Santa Cruz Biotechnology) antibodies. The bound antibodies were detected using horseradish peroxidase-conjugated secondary antibodies and an ECL (Bio-Rad) Western blotting detection reagent. The relative protein levels were normalized to those of β-actin (MA5–15739, Thermo Fisher Scientific, Waltham, MA, USA) used as a loading control.
3
Western Blot Analysis of PEDV-S
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Western blot assays were carried out according to the published procedure with some modifications [35 (link)]. Transfected cells were collected and lysed in 200 μl of mammalian cell lysis buffer (50 mM Tris [pH 8.0], 5 mM EDTA, 100 mM NaCl, 1% NP-40 and protease inhibitor mixture) for 30 min on ice. After centrifugation at 10,000 × g for 5 min, lysates were separated on 10% SDS-PAGE gels and subsequently transferred to nitrocellulose membranes (Bio-Rad), followed by blocking with 5% non-fat milk in TBS-T for 1 h. Membranes were probed with one of the primary antibodies, including anti-PEDV-S mouse polyclonal antibody (a kind gift from Dr. Qigai He), and anti-β-actin mouse monoclonal antibody clone C-4 (Santa Cruz Biotechnology) followed by goat anti-mouse antibodies conjugated to HRP (Biolegend). The signals were visualized with western blotting detection reagent (Bio-Rad).
4
Melanogenesis Regulation Assay
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Resazurin from Sigma (Saint Louis, MO, USA); RPMI-1640 and FCS from PAA; β-actin and anti-rabbit IgG and HRP linked antibody from Cell Signaling technology (Boston, USA); Tyrosinase (H-109) and MITF (H-50) rabbit polyclonal antibody from Santa Cruz Biotechnology, Inc. (Dallas, Texas 75220 USA) and Western blotting detection reagent from Bio-RaD (USA); α-melanocyte stimulating hormone, 3,4-dihydroxy-L-phenylalanine, mushroom tyrosinase, protease inhibitor cocktail, phosphatase inhibitor cocktail, kojic acid, phenylmethylsulfonyl fluoride and QuantiPro BCA Assay Kit from Sigma (Steinheim, Germany); all solvents as analytical grade were purchased from Dr Mojallali Lab (Tehran, Iran).
5
Western Blot Analysis of Liver Proteins
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The liver tissues from rats were lysed using Tissue Lysis Buffer (Cell Biologics, Chicago, IL, USA). Equal amounts (80 μg/lane) of total protein were dissolved in NuPAGE® LDS sample buffer (Life Technologies, Carlsbad, CA, USA) and were separated in a 10% SDS-polyacrylamide gel. The separated proteins were transferred to a nitrocellulose membrane (Bio-Rad Laboratories, Hercules, CA, USA), which was then blocked for 1 h in a blocking buffer. The membrane was then incubated for 12 h at 4 °C with antibodies recognizing IGF-1, IGFBP-3, JAK2, p-JAK2, STAT5, p-STAT5, and β-actin (Cell Signaling Technology, Danvers, MA, USA, 1:1000). This was followed by incubating the membrane with a secondary antibody (anti-rabbit IgG HRP-linked antibody, 1:5000, Cell Signaling Technology, Danvers, MA, USA) for 1 h at room temperature. The immunoreactive protein bands were detected using chemiluminescence (ECL, Bardhaman, India) Western blotting detection reagents (Bio-Rad, Hercules, CA, USA).
6
Exosome Protein Profiling via Western Blot
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Exosome-like vesicles were lysed in 40 μL of lysis buffer (Promega) containing 1 μL of proteinase inhibitor cocktail (Sigma). The total protein concentration was measured using a Bradford assay containing Coomassie Plus protein reagent (Bio-Rad Laboratories) according to the manufacturer’s specifications. Equivalent amounts of total lysate were subjected to SDS-PAGE using 10% polyacrylamide gels. Proteins were electroblotted to polyvinylidene difluoride membrane (Millipore). The membranes were then blocked and incubated in anti-Annexin A2 (rabbit polyclonal; Abcam), Alpha-enolase (mouse monoclonal; Santa Cruz), Anexin A1 (mouse monoclonal; Abcam), and EpCAM (mouse monoclonal; Abcam). Alkaline phosphatase–conjugated anti-mouse or anti-rabbit IgGs were used as secondary antibodies (Bio-Rad) for detection. Then the membranes were incubated with Western Blotting Detection Reagents (Bio-Rad) according to the manufacturer’s instructions and exposed to autoradiography film.
7
Synergistic Effect of SC NPs on 4T1 Cells
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4T1 cells (5×106) in 150 mm dishes were cultured at 37°C for 12 h. The cells were cultured for another 12 h under hypoxic or normoxic conditions. The cells were treated with different formulations, including Srf, CNO, SC Sol, and SC NPs (8 μM Srf and 4 μM CNO) for 6 h. The cell lysates (containing 20 ng protein) were subjected to standard electrophoresis, followed by antibody incubation at 4°C. The dilution ratios for the primary antibody were 1:50000 (β-actin-specific antibody), 1:500 (GPX4-specific antibody), and 1:500 (GR-specific antibody). The dilution ratio of the secondary antibody was 1:5000 for all samples. The protein bands were detected using ECL western blotting detection reagents and monitored using Bio-Rad ChemiDoc. To further verify the synergistic mechanism of SC NPs, 3D tumor spheroids were treated with Srf, CNO, SC Sol, and SC NPs (8 μM Srf and 4 μM CNO) for 48 h. The spheroids were washed with PBS and stained with Calcein-AM (4.5 μM) for 1 h, and stained with PI (2 μM) for 30 min. Spheroids were further detected using z stack imaging at 40-μm intervals from the top of the spheroids through CLSM (ex: 488 nm, em: 515 nm and 617 nm).
8
Protein Extraction and Western Blotting
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Cells were lysed in denaturing lysis buffer containing protease inhibitors (RIPA, Solarbio, Beijing, China) for 30 min on ice and centrifuged (12,000 rpm) for 10 min at 4°C. Protein concentrations in the lysates were determined by a BCA protein assay kit (Solarbio, Beijing, China). Approximately 30 μg of protein was separated by 12% SDS-PAGE and transferred to a nitrocellulose filter membrane that was blocked with 5% nonfat dried milk in Tris-buffered saline containing 0.05% Tween 20 (pH 7.6) for 1 h at 25°C. Subsequently, the membranes were incubated overnight at 4°C with primary antibodies. Then, the blots were incubated with secondary antibodies for 1 h at room temperature. After washing three times with TBST, immunostaining was visualized using Western blotting detection reagents (Bio-Rad, United States). Antibody details are listed in the Supplemental Material .
9
Cardiac Tissue Protein Extraction and Analysis
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Total protein was extracted from cardiac tissues using radioimmunoprecipitation assay (RIPA) buffer, including a protease inhibitor cocktail. A 12% SDS polyacrylamide gel electrophoresis (SDS-PAGE) was used to separate the proteins. Then, the proteins were transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Burlington, MA, USA), which were incubated overnight at 4 °C. Primary antibodies against BAD, AKT, and p-AKT were obtained from Cell Signaling Technology (Danvers, MA, USA), while Bcl-2, Bcl-xl, and GAPDH were purchased from Abcam (Cambridge, MA, USA). Then, secondary antibodies (Abcam, Cambridge, MA, USA) were incubated at 25 °C for 1 h. Bands were visualized using enhanced chemiluminescence (ECL) plus Western blotting detection reagents (Bio-Rad, Hercules, CA, USA) and subsequently captured by the ChemiDoc Touch Imaging System and Image Lab software (Bio-Rad, Hercules, CA, USA).
10
Western Blot Analysis of Apoptosis Regulators
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Total protein was extracted from cells using the RIPA lysis buffer (H1107020, Macgene) according to manufacturer's protocols. An equal amount of proteins was loaded in each lane. Proteins were separated on 12% gradient Tris-glycine gels (10 μl/well, 10 wells) (Sigma Aldrich), and electrically transferred to a PVDF membrane (Bio-Rad). After blocking the membrane with 5% skim milk, the target proteins were immune-blotted with their corresponding antibody at 1:1000 dilution after overnight incubation at 4 °C. Antibodies used were GPX4 (ab125066, Abcam), cleaved caspase-3 antibody (ab2302, Abcam), ACSL4 (22401-1-AP, Proteintech) and beta-actin (ab8226, Abcam). Thereafter, the horseradish peroxidase (HRP)-conjugated anti-rabbit IgG H&L (E030120, Earthox, 1:2000 dilution) and anti-mouse IgG H&L (E030110, Earthox, 1:2000 dilution) were applied as the secondary antibodies, and bands were detected with western blotting detection reagents (Bio-rad) using ECL (S4904300, YEASEN).
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