Whole brains were collected from male hKO and WT mice (n = 4 of each genotype) and mushed and lysed in radioimmunoprecipitation assay (RIPA) buffer containing protease inhibitors. Protein concentration in each sample lysate was adjusted to 0.5 μg/μL with 2× sample buffer. An equal amount volume of each lysate (10 μL) containing 5 µg protein was loaded onto Extra PAGE One precast gels (7.5%, Nacalai Tesque, Kyoto, Japan). Proteins were transferred to a polyvinylidene difluoride (PVDF) membrane using WSE-7210 EzFastBlot HMW (Atto). The membrane was blocked with 5% ECL Prime blocking agent (GE) and incubated with primary antibody, anti-ARID1B (ab57461, abcam) or anti-TUBULIN (PM054, MBL), followed by secondary antibody, either anti-Mouse IgG HRP (#7076, CST) or anti-Rabbit IgG HRP (NA934VS, GE), respectively. The bands were visualized with ECL Prime (GE). Band intensities were quantified by densitometry using the ImageJ software and the quantity of ARID1B protein in each sample was normalized to that of TUBULIN.
Ecl prime
Manufactured by GE Healthcare
Sourced in United Kingdom, United States, Japan, Sweden, France, Germany, Italy, Denmark
ECL Prime is a chemiluminescent detection reagent designed for Western blotting applications. It is used to detect and visualize target proteins that have been separated by gel electrophoresis and transferred to a membrane.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (34 mentions)
Bca protein assay, by Thermo Fisher Scientific (22 mentions)
Immobilon p, by Merck Group (20 mentions)
Ripa buffer, by Thermo Fisher Scientific (20 mentions)
β actin, by Merck Group (18 mentions)
Nitrocellulose membrane, by Bio-Rad (18 mentions)
Protease inhibitor cocktail, by Roche (16 mentions)
Pvdf membrane, by Bio-Rad (15 mentions)
Pvdf membrane, by GE Healthcare (14 mentions)
Imagequant las 4000, by GE Healthcare (13 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (13 mentions)
Enhanced chemi luminescence (ecl), by GE Healthcare (12 mentions)
β actin, by Cell Signaling Technology (12 mentions)
Chemidoc xr, by Bio-Rad (11 mentions)
Protease inhibitor cocktail, by Merck Group (11 mentions)
Nupage bis tris gel, by Thermo Fisher Scientific (11 mentions)
Trans blot turbo transfer system, by Bio-Rad (10 mentions)
Ripa buffer, by Merck Group (10 mentions)
Immobilon p membrane, by Merck Group (10 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (10 mentions)
553 protocols using ecl prime
1
ARID1B Protein Expression in Mice
2
Western Blot Analysis of Cell Signaling Proteins
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Cells were lysed in RIPA buffer (50 mM Tris-HCl buffer, pH 8.0, 150 mM NaCl, 1 % NP-40, 0.5 % deoxycholate, 0.1 % SDS, protease inhibitor cocktail (Roche Diagnostics, Basel, Switzerland), phosphatase inhibitor cocktail (Roche Diagnostics)). Each 10 μg sample of protein was subjected to SDS-PAGE (5-20 % gradient SDS-polyacrylamide gels, BIOCRAFT, Tokyo, Japan), and separated proteins were transferred to polyvinylidene fluoride membrane (HybondTM-P, GE Healthcare). The membranes were incubated with primary antibodies, followed by appropriate secondary antibodies, and then visualized using ECL prime (GE Healthcare). For dot-blot analysis, cell lysate samples (each 2 or 4 μg/spot) were loaded on a nitrocellulose membrane. The membranes were incubated with primary antibodies, followed by appropriate secondary antibodies, and then visualized using ECL prime (GE Healthcare). The images were acquired on LAS 4000 (GE Healthcare). The intensity of the protein band was analyzed using Fiji (http://fiji.sc/ ). The following primary antibodies were used: N-cadherin (1:1,000, Santa Cruz Biotechnology), GAPDH (1:3,000, Abcam), 4E-BP1 (1:1,000, Cell Signaling Technology (CST), Danvers, MA), Phospho-4E-BP1 (Ser65) (1:1,000, CST), eIF4B (1:1,000, CST), Phospho-eIF4B (Ser406) (1:1,000, CST), eIF4E (1:1,000, Abcam), and Phospho-eIF4E (Ser209) (1:1,000, Abcam).
3
Immunoblotting protocol for protein detection
4
Immunoblotting Protein Detection Protocol
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For immunoblotting, protein lysates or immunoprecipitates were denatured by boiling in Laemmle sample buffer, fractionated by SDS-PAGE, and transferred to nitrocellulose membranes. Blots were blocked for 1 h at room temperature with 5% non-fat dry milk in Tris-buffered saline with 0.1% Tween 20 (TBST) for anti-tagBFP, -GAPDH and -G3BP1 antibodies; 5% BSA in TBST was used for blocking anti-G3BP1PS149 antibody. Primary antibodies diluted in appropriate blocking buffer were added to the membranes and incubated overnight incubation at 4 °C with rocking. Primary antibodies consisted of: rabbit anti-G3BP1 (1:2000; Sigma), rabbit anti-G3BP1PS149 (1:1000; Sigma), rabbit anti-TagBFP (1:2000; Evrogen), and rabbit anti-GAPDH (1;2,000; CST). After washing in TBST, blots were incubated HRP-conjugated anti-rabbit IgG antibodies (1:5000; Jackson lab) diluted in blocking buffer for 1 h at room temperature. After washing signals were detected using ECL PrimeTM(GE Healthcare).
5
Rad53 Phosphorylation Analysis
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Wildtype and pds5-1 mutant strains were grown to 0.1–0.3 OD600, arrested in pre-anaphase (nocodazole), pelleted by centrifugation, resuspended in water, and immediately frozen in liquid nitrogen. Cells were mechanically lysed (Bead-beater, BioSpec) in the presence of Trichloroacetic acid (TCA). The precipitated extracts were then solubilized in 4X Laemelli loading buffer (Amresco) and resolved by SDS-PAGE prior to transfer to PVDF membrane. Western blot analysis to assess the level of Rad53 modification was performed using Goat-anti-Rad53 (Santa Cruz, yC-19), Donkey-anti-Goat HRP secondary and signal detection performed following ECL Prime (GE) manufacturer instructions.
6
Subcellular Fractionation and Protein Analysis
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Bacterial cells and the subcellular fractions were subjected to SDS-polyacrylamide gel electrophoresis (SDS–PAGE) followed by an appropriate visualization using a Coomassie brilliant blue (CBB) staining kit (Quick-CBB, Wako Co., Ltd., Osaka, Japan) or a silver staining kit (2D-Silver Stain Reagent II, Cosmo Bio Co., Ltd., Tokyo, Japan), according to the manufacturers’ instructions. Western blot analysis was carried out by standard methods. Rabbit antisera against E. coli FliC (Westerlund-Wikstrom et al., 1997 (link)) and Ag43 (Beloin et al., 2006 (link)) were used as the primary antibodies for Western blot. We also used rabbit antisera against the following subcellular marker proteins: DsbA (localized at periplasm; our collection), Crp (localized at cytoplasm; our collection), OmpC (localized at outer membrane; our collection), and RodZ (localized at inner membrane; purchased from NBRP, NIG, Japan). Horseradish peroxidase (HRP)-labeled anti-rabbit Ig antibody was used as the secondary antibody following these first antibodies. Chemiluminescence was developed by ECL Prime (GE Healthcare Bio-Sciences) or Immobilon ECL Ultra (Millipore, Darmstadt, Germany).
7
Confirming Antibody 0614 Antigen by Western Blot
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The antigen of antibody 0614 was confirmed by Western blotting. Recombinant CD73 with a C-terminal His-tag was loaded onto SDS-PAGE gel, separated, and then transferred to PVDF membranes (Bio-Rad). Membranes were blocked for 5 min with Bullet Blocking One for Western blotting (Nacalai Tesque). The membranes were washed three times with TBS-T. The blots were then incubated with antibody 0614 (10 µg/mL) or anti-His tag antibody (1:10,000, ProteinTech, Rosemont, IL, USA) at room temperature for 1 h. The membranes were washed three times with TBS-T and then incubated with HRP-conjugated anti-rat IgG (1:5000; Jackson Immuno Research, West Grove, PA, USA) or anti-mouse IgG (1:5000; CST, Danvers, MA, USA) at room temperature for 1 h. Can Get Signal solution (Toyobo, Osaka, Japan) was used to reduce background noise. After the membranes were washed three times with TBS-T, proteins were visualized using ECL prime (GE Healthcare).
8
Liver Protein Extraction and Western Blot
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Protein was extracted from frozen liver with lysis buffer until homogenous, and then centrifuged at 13000 rpm at 4°C for 10 min. The supernatant was collected and protein concentration measured using the BCA assay with an ELISA micro-plate reader. Approximately 20 μg of liver protein was separated by SDS–PAGE and the resolved proteins were transferred onto a polyvinylidene difluoride membrane (PVDF). The membranes were blocked and incubated with primary antibody (α–SMA, 1:1000, ab7817, Abcam): diluted in 5% BSA) at 4°C overnight using gentle shaking. Afterwards, the membrane was washed with Tris-buffered saline with 0.1% Tween 20 detergent buffer and incubated with a secondary antibody 1:3000 dilution of HRP-conjugated sheep anti-mouse IgG (Jackson ImmunoResearch) for 1 h. An enhanced chemiluminescence detection reagent (ECL Prime, GE Healthcare) was added onto the membrane and then the developed immunoreactive band was visualized using an Image Quant LAS4000 mini machine (GE Healthcare Bio-Sciences AB, Uppsala, Sweden). Relative band intensity was measured using ImageJ software (National Institutes of Health, Bethesda, MD, USA).
9
Rac1 Activity Assessment in Chondrocytes
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To evaluate Rac1 activity, a Rac1 Pull-down Activation Assay Biochem Kit (Cytoskeleton) was employed. Mouse primary chondrocytes were plated at a density of 1 × 105 cells in 2 × 2 cm stretch chambers. After cyclic tensile strain (0.5 Hz, 10% elongation) for 30 min, proteins were isolated from cells and 500 µg was used in a pull-down assay with p21-activated kinase–Rac1 p21 binding domain beads. The resulting pull-down product was then immunoblotted with Rac1 antibody according to the manufacturer’s instructions. His-tagged Rac1 protein in the kit was used for control. The membranes were incubated with a horseradish peroxidase-conjugated antibody (Promega), and visualized with ECL prime (GE Healthcare) and an AE-6981 Light Capture II (ATTO, Tokyo, Japan). Control cells were seeded onto identical chambers and cultured without cyclic tensile strain. Original images of the assay were shown in Supplementary Fig. 15 .
10
Western Blot Protein Analysis Protocol
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The total cellular samples were washed two times with ice-cold PBS, scraped with IP lysis buffer, and then transferred into a micro centrifuge tube. The cell suspension was maintained with constant agitation for 30 minutes at 4°C. Then the cell lysate was centrifuged at 21 000 × g for 30 minutes at 4°C and supernatants were collected for analysis. The protein concentration was determined using BCA Protein Assay Reagent (Pierce, Rockford, IL, USA).
Samples were boiled for 5 minutes in Laemmli sample buffer containing 2.5% β-mercaptoethanol and 10 μg of sample proteins were separated by 12% TGX Stain-Free polyacrylamide gels (Cat#1610185, Bio-Rad), and transferred to PVDF membrane by Trans-Blot Turbo Transfer System (Bio-Rad). Membranes were blocked with 5% (w/v) fat-free dry milk in Tris-buffered saline (10 mM Tris-HCl, pH 7.5, 150 mM NaCl) containing .05% Tween-20 for 1 hour and incubated with primary antibodies overnight at 4°C. Blots were washed 3 times in TBS buffer and then incubated with appropriate secondary antibodies for 1 hour at room temperature. The protein bands were visualized with the enhanced chemiluminescence reagent (ECL Prime, GE Healthcare, Cat# RPN2232) by an imager (ChemiDoc MP, imaging system, Bio-Rad).
Samples were boiled for 5 minutes in Laemmli sample buffer containing 2.5% β-mercaptoethanol and 10 μg of sample proteins were separated by 12% TGX Stain-Free polyacrylamide gels (Cat#1610185, Bio-Rad), and transferred to PVDF membrane by Trans-Blot Turbo Transfer System (Bio-Rad). Membranes were blocked with 5% (w/v) fat-free dry milk in Tris-buffered saline (10 mM Tris-HCl, pH 7.5, 150 mM NaCl) containing .05% Tween-20 for 1 hour and incubated with primary antibodies overnight at 4°C. Blots were washed 3 times in TBS buffer and then incubated with appropriate secondary antibodies for 1 hour at room temperature. The protein bands were visualized with the enhanced chemiluminescence reagent (ECL Prime, GE Healthcare, Cat# RPN2232) by an imager (ChemiDoc MP, imaging system, Bio-Rad).
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