Ten to fifteen seconds of sonication in 80 µL of lysis buffer [phenylmethanesulfonylfluoride (PMSF), 1 mM; Na3VO4 2 mM; NaF, 100 mM; 1× protease inhibitor cocktail (P8340, Sigma-Aldrich, St. Louis, MO, USA); and RIPA buffer (R0278, Sigma-Aldrich, St. Louis, MO, USA)] were used for retinal protein extraction. Extracted protein (25 µg) was loaded in 4–20% (v/v) mini-PROTEAN TGX precast protein gels (4561096, Bio-rad, Hercules, CA, USA). Electrophoresis was carried out for 90 min at 100 V. Proteins were then transferred from gradient gels to a polyvinylidene difluoride (PVDF) membrane (1620177, Bio-Rad Laboratories, Hercules, CA, USA) for 90 min at 400 mA. Membranes were then blocked in 5% bovine serum albumin (A3059-100G, Sigma-Aldrich, St. Louis, MO, USA) or 5% powdered milk (Central Lechera Asturiana, Spain). Both blocking solutions were prepared in 0.1% TBS-Tween. Primary antibodies (Table 2 ) were applied overnight at 4 °C. Secondary antibodies [goat anti-rabbit and goat anti-mouse (Dako Agilent, Santa Clara, CA, USA)] were incubated (1:10,000). Immunoreactive bands were acquired using a WesternBright ECL HRP substrate kit (K-12045-D50, Advansta, CA, USA) and quantified with Image J software (version 1.53. U. S. National Institutes of Health, Bethesda, MD, USA). Vinculin protein was assigned as loading control to normalize protein expression.
Westernbright ecl hrp substrate kit
Manufactured by Advansta
Sourced in United States
The WesternBright ECL HRP substrate kit is a chemiluminescent detection system used for the visualization of western blot protein bands. The kit contains the necessary reagents for the detection of horseradish peroxidase (HRP)-conjugated secondary antibodies.
Automatically generated - may contain errors
Lab products found in correlation
Bca kit, by Thermo Fisher Scientific (12 mentions)
Pvdf membrane, by Merck Group (5 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (4 mentions)
Ripa buffer, by Merck Group (3 mentions)
Bolt 4 12 bis tris plus gel, by Thermo Fisher Scientific (3 mentions)
Bolt mes sds running buffer, by Thermo Fisher Scientific (3 mentions)
Iblot 2 nitrocellulose transfer stacks, by Thermo Fisher Scientific (3 mentions)
Protease phosphatase inhibitor cocktail, by Cell Signaling Technology (3 mentions)
Uvp chemidoc itts2 imager, by Analytik Jena (3 mentions)
Bca reagent, by Thermo Fisher Scientific (3 mentions)
Protease inhibitor cocktail, by Merck Group (2 mentions)
Pvdf membrane, by Bio-Rad (2 mentions)
Nitrocellulose membrane, by Bio-Rad (2 mentions)
Bhlhe40, by Thermo Fisher Scientific (2 mentions)
Bhlhe41, by Biorbyt (2 mentions)
Ne per kit, by Thermo Fisher Scientific (2 mentions)
Abca1, by Abcam (2 mentions)
Actin, by Merck Group (2 mentions)
Mops running buffer, by Thermo Fisher Scientific (2 mentions)
Precision protein streptactin hrp, by Bio-Rad (2 mentions)
24 protocols using westernbright ecl hrp substrate kit
1
Retinal Protein Extraction and Western Blot
2
Protein Expression Analysis of Liver Tissue
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Liver tissue was lysed with ice-cold PRO-PREPTM protein extract solution (iNtRON; Sungnam, Gyunggi, Korea), and the concentration of the protein was determined by the BCA reagent (Thermo Scientific, Sunnyvale, CA, USA). Equal amounts of protein were separated by SDS-PAGE and transferred onto a nitrocellulose membrane (Bio-Rad, Hercules, CA, USA). The membranes were incubated with TBS-T containing 5% milk and the primary antibodies against MAT I/III, GCLC, α-tubulin (Santa Cruz Biotechnology, Santa Cruz, CA, USA), and CDO (Abcam, Cambridge, MA, USA). After washing with TBS-T, the membrane was incubated with the appropriate horseradish peroxidase-conjugated secondary antibodies. The antigen was detected using a Western Bright ECL HRP substrate kit (Advansta, Menlo Park, CA, USA).
3
Protein Extraction and Western Blotting
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Protein extraction and western blotting was performed as previously described.26 PDLCs were starved for 24 hours, then stimulated by SrCl2, collected with PBS, mixed with loading buffer and then heated at 95°C for 10 minutes for total protein denaturation. Nuclear and cytoplasmic proteins were extracted by Nuclear and Cytoplasmic Protein Extraction Kit (Beyotime Biotechnology, China). The protein contents were determined by using a bicinchoninic acid (BCA) assay kit (Thermo Scientific).
Proteins were separated by SDS‐PAGE then transferred to nitrocellulose membranes. The membranes were blocked using 5% non‐fat milk or 5% BSA for 60 minutes at room temperature. Membranes were incubated with primary antibodies against hnRNPL (1:1000), Setd2 (1:1000), H3K36me3, P‐AKT (1:2000), AKT(1:1000), P‐CREB (1:2000) at 4°C overnight, followed by secondary antibodies for 1 hours at room temperature. Protein bands on the membranes were visualized using Western Bright ECL HRP substrate Kit (Advansta).
Proteins were separated by SDS‐PAGE then transferred to nitrocellulose membranes. The membranes were blocked using 5% non‐fat milk or 5% BSA for 60 minutes at room temperature. Membranes were incubated with primary antibodies against hnRNPL (1:1000), Setd2 (1:1000), H3K36me3, P‐AKT (1:2000), AKT(1:1000), P‐CREB (1:2000) at 4°C overnight, followed by secondary antibodies for 1 hours at room temperature. Protein bands on the membranes were visualized using Western Bright ECL HRP substrate Kit (Advansta).
4
Liver Protein Extraction and Western Blot Analysis
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Western blotting was accomplished as reported previously [23 (link)]. Liver was lysed with ice-cold protein extract solution (iNtRON; Sungnam, Gyunggi, Korea), and the protein concentration was examined by the BCA reagent (Thermo Scientific, Sunnyvale, CA, USA). Equal amounts of protein were separated by SDS-PAGE and transferred onto a nitrocellulose membrane (Bio-Rad, Hercules, CA, USA). The membranes were incubated with TBS-T containing 5% milk and the primary antibodies against CYP2E1 (Detroit R&D, Detroit, MI, USA), GAPDH (Santa Cruz Biotechnology, Santa Cruz, CA, USA). After washing, the membrane was incubated with the horseradish peroxidase-conjugated secondary antibodies. The antigen was detected using a Western Bright ECL HRP substrate kit (Advansta, Menlo Park, CA, USA).
5
Nuclear and Cytoplasmic Protein Fractionation
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Cells were lysed in NE-PER kit to extract nuclear and cytoplasmic fractions separately (Thermo Fisher Scientific, 78833) supplemented with Protease/Phosphatase Inhibitor Cocktail (Cell Signaling, 5872) following manufacturer’s instructions. Protein concentration was measured using BCA kit (Thermo Fisher Scientific, 23225) and equal quantities were used to prepare samples for western blotting. Samples were resolved by electrophoresis with Bolt 4–12% Bis-Tris Plus Gels (Invitrogen) in Bolt MES SDS running buffer (Invitrogen, B0002) and transferred using iBlot 2 nitrocellulose transfer stacks (Invitrogen). Membranes were blocked for 1 h and probed with antibodies: BHLHE40 1:500 (Thermo Fisher Scientific, PA5-83044), BHLHE41 1:500 (Biorbyt, orb224120), APOE 1:1000 (Millipore, AB947), ABCA1 1:1000 (Abcam, 018180), ACTIN 1:10,000 (Sigma-Aldrich, A5441) in 5% non-fat dry milk in PBS/0.1% Tween-20 buffer overnight at 4 °C. Secondary antibody staining 1:10000 was applied for 1 h at RT, visualized using WesternBright ECL HRP Substrate Kit (Advansta, K-12045), and measured using iBrigh imagining system (Applied Bioscience). Images were analyzed using ImageJ (NIH). Uncropped western blot images are pasted in Source Data .
6
Western Blot Protein Detection
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Cultured cells were lysed in RIPA buffer. Protein concentration in each sample was determined using Pierce BCA protein assay kit (23227, ThermoFisher). RIPA buffer, 2-Mercaptoethanol (BME), and 6x gel loading dye were added to the protein lysate to create gel ready samples at 1 μg/μl. Samples were heated at 70°C for 10 min to reduce protein. Protein was loaded on an 8% Bis-Tris Plus Gel (NW00082, Thermo Fisher) and electrophoresed in MOPS running buffer (B001, Invitrogen) at 200 V for 35 min. Protein was transferred onto 0.45 μm nitrocellulose membrane in Tris-Glycine-Methanol buffer at 250 mA for 1 h. The transferred blot was blocked in 5% (w/v) non-fat milk in Tris-Buffered Saline with Tween 20 (TBS-T) for 60–90 min at room temperature. Blots were incubated with primary anti-TRPC6 (LS-C19628) or anti-eGFP (AB6556) antibodies in blocking solution @1:1000 dilution and left overnight at 4°C. After washing in TBS-T, goat anti-rabbit secondary antibody was applied at 1:50 k in TBS-T, while Precision Protein StrepTactin-HRP (1610380, BioRad) was applied at 1:100 k to stain for the molecular weight ladder. Blots were incubated in WesternBright ECL HRP substrate kit (K012045, Advansta, San Jose, CA, USA) for 2 min and imaged on Bio-Rad Image (Bio-Rad).
7
Synaptic Protein Extraction and Analysis
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Synaptosome protein samples were extracted following the manufacturer's protocols (Syn‐PER™ Synaptic Protein Extraction Reagent, 87793; Thermo Fisher). Equal amounts of protein samples were loaded and separated by 4%–12% SDS–PAGE (GenScript ExpressPlus™ PAGE Gel, M00653) and transferred to PVDF membranes (Millipore). Membranes were incubated with primary antibody overnight at 4°C. After washing, the membranes were incubated with peroxidase‐conjugated secondary antibodies (1:5000, goat anti‐rabbit IgG, goat anti‐mouse IgG, Abcam) for 1.5 h at room temperature. The immunoreactive bands were visualized by chemiluminescent HRP substrate (WesternBright ECL HRP Substrate Kit, K‐12045‐D50‐EA, Advansta) with a Fluorchem E Chemiluminescence Gel Imaging System (Protein Simple). The intensity of the immunoreactive different exposure times for the same strip was determined with ImageJ software. The expression levels of the proteins of interest were examined as GAPDH/total protein in a semiquantitative manner (see Supporting Information ).
8
Quantification of Amyloid-Beta Peptides
9
Quantitative Western Blot Analysis of Glomerular Proteins
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Total proteins were extracted from glomerulus by lysis buffer and the protein concentration was detected by using the BCA assay. The final concentration of protein samples was diluted to 10 mg/ml mixed with loading buffer. A total of 10 μL protein sample was added to the prepared gels and separated by SDS-PAGE. The proteins were then transferred to a PVDF membrane (0.45 μm, Millipore, United States), followed by blocking in 5% skim milk powder for 2 h at room temperature. The membranes were incubated at 4°C overnight with the primary antibodies against mTOR, phospho-mTOR, Akt, phospho-Akt, Bax, Bcl-2, caspase-3, β-actin, and GAPDH (diluted at 1:1000). Next, the membrane was washed by PBST and incubated with goat anti-rabbit IgG antibody (Diluted at 1:100000) or goat anti-mouse IgG antibody (Diluted at 1:10000) for 2 h at room temperature. Proteins were assessed by WesternBright ECL HRP substrate kit (Advansta, 171005-80, United States) in a chemiluminescence system (CLiNX ChemiScope 6000 Touch, Shanghai, China).
10
Western Blot Protein Analysis Protocol
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Cells or tumor tissues were lysed using lysis buffer (20 mM Tris-HCl, 1 mM EDTA, 1 mM ethylene glycol tetraacetic acid,10 mM NaCl, 1 mM Na3VO4, 0.1 mM phenylmethylsulfonyl fluoride, 25 mM β-glycerol phosphate, 30 mM sodium pyrophosphate, and 1% Triton X-100; pH 7.4). Lysates were separated using sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred onto nitrocellulose or polyvinylidene difluoride membranes. After blocking with 5% nonfat milk, membranes were immunoblotted with various primary antibodies. The bound antibodies were visualized with horseradish peroxidase (HRP)-conjugated secondary antibodies using an Advansta Western Bright ECL HRP Substrate Kit (Advansta Inc., San Jose, CA, USA).
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