Hepatic Plin2 protein levels were determined by immunoblotting. 20 mg of liver tissue were homogenized in lysis buffer containing 1% NP-40, 0.5% Triton, 10% glycerol, 0.15 M NaCl, 0.001 M EDTA, 0.5 M Tris·HCl, at pH 7.4, and supplemented with complete protein inhibition cocktail tablet from Roche (Penzberg, Germany). 30 mcg of protein were separated by 4–12% NuPage Bis-Tris gel (Invitrogen, Grand Island, NY) and wet transferred to nitrocellulose membranes overnight at 4°C. Membranes were blocked with 5% milk for 1 hour at room temperature and incubated with guinea pig polyclonal antibody against Plin2 (Fitzgerald, Concord, MA) at 1∶1000 dilution overnight at 4C. After serial washes, horseradish peroxidase-conjugated goat anti-guinea pig secondary antibody was applied at 1∶5000 dillution at room temperature for one hour followed by washing and visualization with enhanced chemiluminescence (GE Healthcare, Piscataway, NJ). Membranes were stripped and blotted for GAPDH at 1∶1000 dilution (Cell Signaling, Beverly, MA) [19] .
Enhanced chemiluminescence
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Enhanced chemiluminescence is a detection method used in molecular biology and biochemistry to quantify the presence of specific proteins or other biomolecules. It relies on the emission of light produced by a chemical reaction catalyzed by an enzyme or other agent. The intensity of the light is proportional to the amount of the target molecule, allowing for its sensitive and accurate measurement.
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Lab products found in correlation
Pvdf membrane, by Merck Group (26 mentions)
Nitrocellulose membrane, by Bio-Rad (19 mentions)
Complete protease inhibitor cocktail, by Roche (16 mentions)
Ripa buffer, by Thermo Fisher Scientific (16 mentions)
β actin, by Merck Group (14 mentions)
β actin, by Cell Signaling Technology (14 mentions)
Protease inhibitor cocktail, by Merck Group (14 mentions)
Polyvinylidene difluoride membrane, by Merck Group (14 mentions)
Gapdh, by Merck Group (13 mentions)
Nitrocellulose membrane, by GE Healthcare (11 mentions)
Immobilon p membrane, by Merck Group (11 mentions)
Image lab software, by Bio-Rad (10 mentions)
Protease inhibitor cocktail, by Roche (10 mentions)
Protease inhibitor, by Roche (10 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (10 mentions)
Gapdh, by Santa Cruz Biotechnology (9 mentions)
Pro prep protein extraction solution, by iNtRON Biotechnology (9 mentions)
Anti β actin, by Merck Group (9 mentions)
Protein assay reagent, by Bio-Rad (9 mentions)
Pvdf membrane, by Bio-Rad (9 mentions)
612 protocols using enhanced chemiluminescence
1
Quantitative Analysis of Hepatic Plin2 Protein
2
Adenoviral Protein Expression Analysis
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Protein lysates were collected in ice-cold lysis buffer (150 mM NaCl, 50 mM Tris, 0.05% SDS, 1% triton) and quantified using a Pierce BCA assay kit (Thermo Scientific). Protein samples (20–30 µg) underwent routine electrophoresis on precast 4–12% Bis TRIS pre-cast gels (Invitrogen) and were transferred to nitrocellulose membranes (GE Healthcare). Blots were developed using enhanced chemiluminescence (GE Healthcare) and imaged with an Amersham Image 600 (GE Healthcare). Densitometry analysis was performed using ImageQuant TL software. E1A: Santa Cruz sc-430, 1:1000; Adenovirus: Abcam ab36851, 1:5000; Hsc70: Abcam ab36851, 1:10000; GAPDH: Santa Cruz sc-47724, 1:10000; phospho-Akt: Cell Signalling 9271L, 1:1000; Akt: Cell Signalling 9272; phospho-ERK1/2: Cell Signalling 4370, 1:1000; ERK1/2: Cell Signalling 9102; ARRB1/2: Cell Signalling 4674S, 1:500; ARRB1: Cell Signalling 12697S, 1:500.
3
Immunoblotting of Synaptic Proteins
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Proteins were transferred from the acrylamide gel onto the nitrocellulose (0.22 μm, GE Healthcare) or PVDF membranes. Membranes were incubated with the primary antibodies (α-TSPAN5 1:500; α-transferrin receptor 1:500; α-tubulin 1:40,000; α-PSD-95 1:1,000; α-synaptophysin 1:500; α-NLG1 1:500; α-GluA2 1:500; α-GluA2/3 1:2,000; α-N-Cadherin 1:500; α-neuroligin-3 1:1,000; α-GFP 1:2,500; α-HA 1:1,000, see Key Resource Table for Catalogue Numbers) at room temperature for 2–3 h or overnight at 4°C in TBS Tween-20 (0.1%), milk (5%). After washing, the blots were incubated at room temperature for 1 h with horseradish peroxidase-conjugated α-rabbit, α-mouse or α-rat antibodies (1:2,000) in TBS Tween-20 (0.1%), milk (5%). Immunoreactive bands on blots were visualized by enhanced chemiluminescence (GE Healthcare).
4
Western Blot Analysis of TLR4 and HMGB1
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Lumbar spinal cord homogenates were resolved on a 10% sodium dodecyl sulphate polyacrylamide gel and electro-transferred onto nitrocellulose membranes. The membrane was incubated with anti-TLR4 (1:500; Santa Cruz Biotechnology, Dallas, Texas, USA) or anti-HMGB1 (1:1000; Abcam, Melbourne, Victoria, Australia) antibodies and were detected with enhanced chemiluminescence (GE Healthcare, Sydney, New South Wales, Australia). Densitometric analyses of immunoreactive bands were quantified as described previously [12 (link)].
5
Western Blot Analysis of Protein Expression
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Forty-eight hours after siRNA transfection, asynchronous cells were lysed in TEN-N buffer (1% NP-40, 100 mM NaCl, 10 mM Tris-HCl, pH 7.5, and 1 mM EDTA). Protein concentration was measured by the Bio-Lad Protein assay kit (Bio-Rad, Hercules, CA, USA). Extracted proteins were boiled for 10 min with 4 × NuPAGE® LDS (lithium dodecyl sulfate) sample buffer (Thermo Fisher Scientific). Proteins were separated by NuPAGE® SDS (sodium dodecyl sulfate) PAGE (polyacrylamide gel electrophoresis)-Gel System (Thermo Fisher Scientific), electroblotted onto a polyvinylidene difluoride membrane (Amersham Hybond-P, GE Healthcare), and reacted with appropriate primary antibodies overnight at 4 °C for immunodetection. Blocking and antibody incubation were performed in 3% non-fat dry milk. Proteins were detected using horseradish peroxidase-labeled secondary antibodies (1:3000, Santa Cruz Biotechnology, Dallas, TX, USA) for 1 h at room temperature and enhanced chemiluminescence, according to the manufacturer’s instructions (GE Healthcare).
6
Western Blotting of LC3b Protein
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Western blotting was performed, as previously described [34 (link)]. Briefly, 50 μg of lysate protein was heated and applied to SDS-PAGE gels, transferred to nitrocellulose membranes, and then immunoblotted. Antibodies used in this study include LC3b (#2775, Cell Signaling) and Rabbit HRP-conjugated secondary antibodies (#7076, Cell Signaling). Visualization was performed with enhanced chemiluminescence (GE Healthcare). The relative band intensities were quantified using ImageJ.
7
Immunoblotting of Cellular Proteins
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Cells were collected in a sodium dodecyl sulfate (SDS) lysis buffer. After sonication, the proteins were subjected to electrophoresis in a 5–20% SDS–polyacrylamide gel, and then transferred onto a polyvinylidene difluoride membrane (ATTO), followed by overnight blocking with 5% skimmed milk in phosphate-buffered saline with Tween 20 (Bio-Rad, Hercules, CA, USA). The membrane was probed with antibodies specific to MAP2K3 (Cell Signaling Technology, Boston, MA, USA), GAPDH (Santa Cruz), and/or β-tubulin (Abcam K.K., Tokyo, Japan). After washing the membrane, it was incubated with secondary horseradish peroxidase-conjugated antibodies for an hour. The signals were detected with enhanced chemiluminescence (GE Healthcare, Tokyo, Japan) and scanned with an image analyzer LAS-4000 mini (Fuji Film, Tokyo, Japan), as previously described [13 (link)].
8
Western Blot Analysis of Lung Tissue
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Western blot analysis was performed as previously4 (link). In brief, lung tissue was collected and protein prepared in FastPrep-24 lysing matrix tubes (MP Biomedicals) then lysed using Radio-Immunoprecipitation Assay (RIPA) buffer (50 mM TricCl pH 7.4, 1% NP40, 0.25% sodium deoxycholate, 150 mM NaCl, 1 mM EDTA) containing protease (Calbiochem, San Diego, CA, USA) and phosphatase inhibitors (Sigma-Aldrich Corp.). 20 μg protein was run on an SDS 8–12% Tris-Glycine gel (Novex, Life Technologies) and then transferred onto a 0.2-μm nitrocellulose membrane (BioRad) overnight, blocked with 1% milk and probed for GR (1:1,000, clone M-20, sc-1004, Santa Cruz), Cav1 (1:1000, clone N20, Santa Cruz), Cavin (1:1000), actin (1:1000), TFIIB (1:1000, Santa Cruz) used to confirm nuclear lysis, Histone 3 (1:1000, Cell Signalling). Immunoreactivity was visualized using enhanced chemiluminescence (GE Healthcare). Densitometry was performed using ImageJ.
9
Protein Extraction and Western Blot Analysis
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Muscle tissue and cell extracts were lysed in and total protein was extracted. Protein concentration was determined by Bradford Protein Assay following manufacturer’s instructions (BioRad). Further, 50 μg of proteins were separated by SDS polyacrylamide gel membranes (PAGE) and transferred to PVDF membranes. After blocking with 5% skim milk, blots were incubated overnight at 4 °C with primary antibodies diluted in 0.1% bovine serum albumin (BSA). After washing, membranes were incubated with horseradish peroxidase-conjugated secondary antibodies for 1 h at room temperature. Blots were visualized by enhanced chemiluminescence (GE Healthcare) and exposed on Chemidoc (BioRad). Quantification was performed using Image Lab software (BioRad) normalizing the values with α-Tubulin.
10
Western Blot Analysis of Apoptosis Markers
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Cells were lysed, and the total protein was obtained using RIPA buffer (Beyotime Institute of Biotechnology). A bicinchoninic acid assay kit (Pierce; Thermo Fisher Scientific, Inc.) was used to quantify the total protein. Equal amounts of protein were separated by 12% SDS-PAGE for 40 min and then transferred to PVDF membranes (EMD Millipore). The membranes were blocked for 1.5 h at room temperature with 5% nonfat milk and incubated with primary antibodies including anti-FA2H (Cat no. ab128917; 1:1,000; Abcam), anticleaved caspase-3 (Cat no. ab32042; 1:1,000; Abcam), and anti-pro-caspase-3 (Cat no. ab32499; 1:1,000; Abcam) overnight at 4°C. The next day, the membranes were incubated with horseradish peroxidase-conjugated antirabbit secondary antibody (Cat. no. 7074; 1:2,000; Cell Signaling Technology, Inc.) for 2 h. Protein bands were visualized by enhanced chemiluminescence (GE Healthcare Life Sciences). β-Actin (1:1,000; Abcam) served as the loading control for normalization.
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