Cells were harvested and lysed in ice-cold M-PER mammalian protein extraction reagent (Pierce, Rockford, IL, USA) containing 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, and protease inhibitor cocktail (Roche Diagnostics GmbH, Mannheim, Germany), and subjected to centrifugation at 10,000 rpm for 10 min. Protein concentrations were determined by BCA protein assay. A 30–40 µg aliquot of protein from each treatment was subjected to 10% SDS-PAGE. After electrophoresis, the separated proteins were transferred to Immun-blotTM PVDF membranes (Bio-Rad) by semi-dry blotting, and probed with the appropriate antibody, followed by incubation with Odyssey secondary antibodies, according to manufacturers’ instructions (goat anti-rabbit IRDye 680 or 800 and goat anti-mouse IRDye 680 or 800, depending on required combinations). Blots were imaged and quantitated using an Odyssey Infrared Imaging System (LI-COR Biosciences, Lincoln, NE, USA).
Immun blot pvdf membrane
Manufactured by Bio-Rad
Sourced in United States, Japan, United Kingdom
The Immun-Blot PVDF membrane is a product designed for Western blotting applications. It is made of polyvinylidene fluoride (PVDF) material and serves as a support surface for protein transfer and immobilization during the Western blotting process.
Automatically generated - may contain errors
Lab products found in correlation
Pierce bca protein assay kit, by Thermo Fisher Scientific (14 mentions)
Clarity western ecl substrate, by Bio-Rad (12 mentions)
Protease inhibitor cocktail, by Merck Group (11 mentions)
Las 4000, by Cytiva (11 mentions)
Ripa buffer, by Thermo Fisher Scientific (9 mentions)
Odyssey fc imaging system, by LI COR (9 mentions)
Nupage lds sample buffer, by Thermo Fisher Scientific (8 mentions)
Supersignal west pico plus chemiluminescent substrate, by Thermo Fisher Scientific (8 mentions)
Supersignal west pico chemiluminescent substrate, by Thermo Fisher Scientific (8 mentions)
Mini protean tgx gel, by Bio-Rad (7 mentions)
Supersignal west dura extended duration substrate, by Thermo Fisher Scientific (7 mentions)
Amersham ecl prime, by Cytiva (7 mentions)
Chemidoc imaging system, by Bio-Rad (7 mentions)
Chemidoc mp imaging system, by Bio-Rad (7 mentions)
Pierce ecl western blotting substrate, by Thermo Fisher Scientific (6 mentions)
Amersham ecl prime western blotting detection reagent, by GE Healthcare (6 mentions)
Dc protein assay kit, by Bio-Rad (6 mentions)
Amersham imager, by Cytiva (6 mentions)
Odyssey blocking buffer, by LI COR (6 mentions)
Ripa buffer, by Merck Group (5 mentions)
299 protocols using immun blot pvdf membrane
1
Protein Extraction and Western Blotting
2
Western Blot Analysis of Synaptic Proteins
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After the PCP or olanzapine treatments, cells were harvested with lysis buffer (containing NP40, Protease Inhibitor Cocktail, 1 mM PMSF and 0.5 mM β-glycerophosphate). Total protein concentrations were determined by DC-Assay (Bio-Rad, Hercules, CA), and detected using a SpectraMax Plus384 absorbance microplate reader (Molecular Devices, Sunnyvale, CA). Samples were heat-treated in Laemmli buffer at 95 °C, loaded to 8% SDS-PAGE gels for fractionation, and then transferred onto Immun-BlotTM PVDF membranes (Bio-Rad, Hercules, CA). The block consists of 5% BSA in TBST. The membranes were then incubated with synaptophysin, PSD95 (Life Technologies, NSW; dilution factor 1:1000), BDNF, NRG1 (Santa Cruz Biotechnology, Santa Cruz, CA; dilution factor 1:500), Akt, p-Akt, GSK3β, and p-GSK3β (Cell Signalling Technology, Beverly, MA; dilution factor 1:1000) antibodies in TBST containing 1% BSA overnight at 4 °C. Secondary antibodies were anti-rabbit IgG conjugated with horseradish peroxidase (Santa Cruz Biotechnology, Santa Cruz, CA; dilution factor 1:5000). For visualization, ECL detection reagents were used and films were exposed on the AGFA CP1000 Tabletop Processor (AGFA Healthcarem Scoresby, VIC). Films were analysed using Quantity One software connected to a GS-690 Imaging Densitometer (Bio-Rad, Hercules, CA).
3
Quantifying Synaptic Protein Expressions
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After electrical stimulation, cells were harvested with lysis buffer (containing NP40, Protease Inhibitor Cocktail, 1 mM PMSF and 0.5 mM β-glycerophosphate). Total protein concentrations were determined by BCA Assay (Thermo Fisher Scientific, NSW), and detected using a SpectraMax Plus384 absorbance microplate reader (Molecular Devices, Sunnyvale, CA). Samples were heat-treated in Laemmli buffer at 95 oC, loaded to 8% SDS-PAGE gels for fractionation, and then transferred onto Immun-BlotTM PVDF membranes (Bio-Rad, Hercules, CA). The block consisted of 5% BSA in TBST. The membranes were then incubated with synaptophysin, PSD95 (Thermo Fisher Scientific, NSW; dilution factor 1:1000), and PSA-NCAM (Thermo Fisher Scientific, NSW; dilution factor 1:1000) antibodies in TBST containing 1% BSA overnight at 4 oC. Secondary antibodies were anti-rabbit IgG conjugated with horseradish peroxidase (Santa Cruz Biotechnology, Santa Cruz, CA; dilution factor 1:5000). For visualization, ECL detection reagents were exposed on the GelDoc System (Bio-Rad, Hercules, CA). Images were analysed using ImageJ 1.46r software (NIH, USA).
4
Protein Extraction and Western Blot Analysis
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After 24 h treatments, cells were harvested with lysis buffer containing NP40 (Sigma-Aldrich), Protease Inhibitor Cocktail (Sigma-Aldrich), 1 mM PMSF (Sigma-Aldrich), and 0.5 mM β-glycerophosphate (Sigma-Aldrich). Total protein concentrations were determined by DC-Assay (Bio-Rad, Sydney) and detected with a SpectraMax Plus384 absorbance microplate reader (Molecular Devices, Sunnyvale, CA). Samples were heat-treated in Laemmli buffer at 95°C, loaded to 10% SDS-PAGE gels (Bio-Rad) for fractionation, and then transferred into Immun-Blot TM PVDF membranes (Bio-Rad). The blocking buffer consisted of 5% slim milk in TBST. The membranes were incubated with NPY (sc-28943, Santa Cruz Biotechnology, Santa Cruz), phospho-GSK3β(Ser9) (#9323s, Cell Signaling Technology), and β-Catenin (#8480s, Cell Signaling Technology) antibodies in TBST containing 1% milk at 4°C overnight. Secondary antibodies were anti-rabbit IgG conjugated with horseradish peroxidase (Santa Cruz Biotechnology). For visualization, we used ECL detection reagents and obtained high resolution images with Amersham Gel Imager (GE Healthcare life Sciences).
5
Quantitative Analysis of OXPHOS Proteins
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Thirty micrograms of whole brain cerebrum or cerebellum extracts was electrophoresed in an SDS-12% PAGE gel, transferred to Immun-Blot™ PVDF membranes (Bio-Rad, Hercules, CA, USA) and probed with MitoProfile® Total OXPHOS Rodent WB Antibody Cocktail of antibodies (MitoSciences, Eugene, OR, USA). Protein–antibody interaction was detected with peroxidase-conjugated mouse anti-mouse IgG antibody (Sigma-Aldrich, St Louis, MO, USA), using Amersham™ ECL Plus western blotting detection system (GE Healthcare Life Sciences, UK). Quantification of proteins was carried out using NIH ImageJ 1.37V software. Average gray value was calculated within selected areas as the sum of the gray values of all the pixels in the selection divided by the number of pixels.
6
Hippocampus Protein Analysis in Rat Model
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SD rats were classified into four groups (control, LIPUS, AlCl3, and LIPUS plus AlCl3), 4 h after the last LIPUS stimulation, six rats were killed and fresh hippocampus tissues were homogenized by T-Per extraction reagent (Pierce Biotechnology, Inc.). Lysates were centrifuged and supernatants were harvested and assayed with Protein Assay Reagent (Bio-Rad, CA, U.S.A.). A 40 μg protein was placed on 4–20% SDS-PAGE, transferred to Immun-Blot® PVDF membranes (Bio-Rad, CA, U.S.A.), blocked for 1 h with tris-buffered saline tween-20 (TBST)-containing 5% (w/v) skimmed milk, and then incubated overnight at 4°C in a solution with the primary antibody. A horseradish peroxidase-conjugated secondary antibody was incubated for 1 h at room temperature. Western Lightning ECL reagent Pro (Bio-Rad, CA, U.S.A.) was used to develop the signals, which was captured by ImageQuant™ LAS 4000 biomolecular imager (GE Healthcare Life Sciences, Pennsylvania, U.S.A.) and quantitated by Image J software (National Institute of Health, Bethesda, MD, U.S.A.).
7
Protein Immunoblotting Technique
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Proteins were resolved by SDS-PAGE and electrophoretically blotted onto Immun-Blot PVDF membranes (Bio-Rad, Hercules, California), followed by blocking in 5% nonfat milk. Primary antibodies (Abs) used included rabbit polyclonal anti-AQP5 (#AQP005, Alomone Labs, Jerusalem, Israel), -p300 (#SC584, Santa Cruz Biotechnology, Dallas, Texas), -HDAC2 (#SC7899, Santa Cruz Biotechnology) and -HDAC3 (SC11417, Santa Cruz Biotechnology). Protein was normalized to eukaryotic initiation factor 2α (eIF-2α), β-actin or GAPDH using rabbit anti-eIF2α (#11386, Santa Cruz Biotechnology) polyclonal Ab and mouse anti-β-actin (LMAB-C4, Seven Hills, Cincinnati, OH) and anti-GAPDH (#AM4300, Applied Biosystems, Foster City, CA) monoclonal Abs, respectively. Blots were incubated with horseradish peroxidase-linked anti-IgG conjugates (Promega) for 45 min at room temperature (RT). Complexes were visualized by enhanced chemiluminescence (ECL) (Thermo Scientific, Waltham, MA) with a FluorChem 8900 Imaging System (Alpha Innotech/Cell Biosciences, Santa Clara, CA).
8
Nuclear Protein Extraction and Western Blot
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Western blot was performed using total proteins and nuclear extracts obtained from approximately 2.0 × 106 cells. Cells were collected following standard procedures. Then, 1.0 mL of ice cold RIPA Lysis buffer (Thermo Scientific, Waltham, MA, USA) was added, with freshly added Protease and Phosphatase Inhibitors Cocktails (Thermo Scientific). SOX2, WWTR1/TAZ and YAP1 were investigated in nuclear fractions, obtained using CelLytic™ NuCLEAR™ EXTRACTION KIT (Sigma-Aldrich). Total proteins and nuclear fractions were measured in the extract by the Bradford assay.
The nuclear fractions and the whole cell lysates were examined on Mini-PROTEAN TGX Gels 4–20% (Bio-Rad). Proteins were transferred from gels on Immun-Blot PVDF Membranes (Bio-Rad) in the transfer buffer (glycine, tris [pH 8.4] and methanol) using Mini Trans-Blot Cell apparatus (Bio-Rad). Membranes containing proteins were blocked with milk 5X (Sigma-Aldrich) in TBST and, subsequently, probed with primary and horseradish peroxidase conjugated secondary antibodies following standard procedures. Proteins transferred membranes were washed with TBST and developed with Pierce ECL Western Blotting Substrate (Thermo Scientific). Details are provided in the onlineSupporting Information .
The nuclear fractions and the whole cell lysates were examined on Mini-PROTEAN TGX Gels 4–20% (Bio-Rad). Proteins were transferred from gels on Immun-Blot PVDF Membranes (Bio-Rad) in the transfer buffer (glycine, tris [pH 8.4] and methanol) using Mini Trans-Blot Cell apparatus (Bio-Rad). Membranes containing proteins were blocked with milk 5X (Sigma-Aldrich) in TBST and, subsequently, probed with primary and horseradish peroxidase conjugated secondary antibodies following standard procedures. Proteins transferred membranes were washed with TBST and developed with Pierce ECL Western Blotting Substrate (Thermo Scientific). Details are provided in the online
9
Immunoblotting Analysis of Apoptosis Regulators
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Total proteins were extracted from cultured cells using RIPA buffer containing protease inhibitor and phosphatase inhibitor (Thermo Fisher Scientific, Waltham, MA, USA). Proteins were subjected to 12% SDS‐PAGE (Bio‐Rad, Hercules, CA, USA). The separated proteins were transferred to Immun‐Blot PVDF membranes (Bio‐Rad) and incubated with anti‐ACTB antibody (A2066; Sigma‐Aldrich; 1:1000 dilution), anti‐cleaved PARP antibody (#5625; Cell Signaling Technology, Beverly, MA, USA; 1:1000 dilution), anti‐p21Waf1/Cip1 antibody (#2947; Cell Signaling Technology; 1:1000 dilution), anti‐Bax antibody (#5023; Cell Signaling Technology, Beverly, MA, USA; 1:1000 dilution), anti‐p53 antibody (Clone DO‐7; DAKO Cytomation, Carpinteria, CA, USA; 1:1000 dilution) and anti‐Noxa antibody (OP180; Calbiochem, Darmstadt, Germany; 1:1000 dilution) at 4°C overnight. Next, the membranes were incubated with HRP‐linked anti‐rabbit IgG (GE Healthcare Biosciences, Piscataway, NJ, USA) and HRP‐linked anti‐mouse IgG (GE Healthcare Biosciences) at a dilution of 1:100 000 for 1 hour at room temperature. The antigen–antibody complex was detected using an ECL Prime Western Blotting Detection Kit (GE Healthcare Biosciences).
10
Protein Extraction and Western Blotting
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Cells were harvested, lysed in 1 ml of ice-cold mammalian protein extraction reagent (M-PER, Pierce, Rockford, IL) containing 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride and protease inhibitor cocktail (Roche Diagnostics GmbH), and centrifuged at 15,000 x g for 10 min. Protein concentrations were determined by BCA protein assay (Pierce); 50 μg aliquots of protein from each treatment were subjected to 10% SDS-PAGE. After electrophoresis, the separated proteins were transferred to Immun-blot™ PVDF membranes (Bio-Rad) by semi-dry blotting and were probed with the appropriate antibody followed by incubation with anti-rabbit (CAT.# RPN4301) or anti-mouse IgG (CAT.# RPN4201) conjugated to horseradish peroxidase (Amersham Biosciences, Piscataway, NJ). Polyclonal PPARα, -β, and -γ and RXRα antibodies were used at 1:500 to 1:1000 dilutions in PBS containing 5% non-fat dry milk. Immunoreactive proteins were detected by using ECL™ western blotting (Amersham Biosciences, Piscataway, NJ).
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