Cultured GMCs were scrapped from flasks and homogenized in ice-cold RIPA buffer supplemented with a protease inhibitor cocktail (Thermo Scientific, Rockford, IL, USA). The proteins were then isolated and separated by 4–20% ExpressPlus PAGE Gels (GenScript, Piscataway, NJ, USA) and transferred onto PVDF membranes using a Semi-Dry Blotter (Thermo Scientific). The membranes were blocked with a 5% BSA blocking buffer for ~1 h at room temperature. The membranes were then probed overnight at 4 °C with respective primary antibodies. After a wash in Tris-buffered saline supplemented with 0.05% Tween 20 (TBST), the membranes were probed with horseradish peroxidase-conjugated secondary antibodies for 45 min at room temperature and washed in TBST. The membranes were then incubated with a chemiluminescence reagent (Thermo Scientific), and the immunoreactive protein bands were visualized and documented using the ChemiDoc imaging system (Bio-Rad Laboratories, Inc., Hercules, CA, USA).
Chemiluminescence reagent
Manufactured by Thermo Fisher Scientific
Sourced in United States, Canada, China
Chemiluminescence reagent is a laboratory product used to detect and measure the presence of specific analytes through a chemiluminescent reaction. It is designed to produce light emission during a chemical reaction, which can be quantified to provide information about the target substance.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (12 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (7 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (6 mentions)
Ripa lysis buffer, by Beyotime (6 mentions)
β actin, by Abcam (6 mentions)
Chemidoc imaging system, by Bio-Rad (5 mentions)
β actin, by Cell Signaling Technology (5 mentions)
Protease inhibitor cocktail, by Thermo Fisher Scientific (4 mentions)
Quantity one software, by Bio-Rad (4 mentions)
Gapdh, by Cell Signaling Technology (4 mentions)
Protease inhibitor cocktail, by Roche (4 mentions)
Bca kit, by Beyotime (4 mentions)
β actin, by Santa Cruz Biotechnology (4 mentions)
Bca protein assay kit, by Abcam (4 mentions)
Gel pro analyzer, by Thermo Fisher Scientific (4 mentions)
Ripa buffer, by Merck Group (3 mentions)
X ray film, by Fujifilm (3 mentions)
Bca protein assay kit, by Beyotime (3 mentions)
Imagequant las 4000, by GE Healthcare (3 mentions)
Las 4000, by Cytiva (3 mentions)
164 protocols using chemiluminescence reagent
1
Protein Extraction and Western Blotting
2
Western Blot Analysis of Neurological Markers
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Brain and spinal cord were collected and lysed in RIPA buffer (Sigma, St. Louis, MO, USA) containing protease inhibitors (Complete Mini, Roche, Basel, Switzerland). The primary antibodies used: anti-beta-actin HRP-conjugated (Cat# HRP-60008, RRID:AB_2819183 Proteintech, Rosemont, IL, USA ), anti-ISL1 (mouse, Cat# 39.4D5, RRID:AB_2314683 Hybridoma Bank, Iowa City, IA, USA), anti-NCALD (rabbit, Cat# 12925-1-AP, RRID:AB_2149410 Proteintech, Rosemont, IL, USA), anti-SMN (mouse, Cat# 610646, RRID:AB_397973 BD Biosciences, San Jose, CA, USA). Signal was detected with rabbit-HRP-conjugated secondary antibody (Cat# 7074, RRID:AB_2099233 Cell Signaling Technology, Danvers, MA, USA) and mouse-HRP-conjugated secondary antibody (α-mouse, Cat# 115-035-003, RRID:AB_10015289 Jackson ImmunoResearch Labs, West Grove, PA, USA) and the chemiluminescence reagent (Thermo Fisher Scientific, Waltham, MA, USA).
3
Protein Expression Analysis of BMSCs
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Total protein was extracted from BMSCs using a mammalian cell extraction kit according to the manufacturer’s instructions (Biovision, Mountain View, CA). Aliquots of twenty micrograms of protein were separated via SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes. The membranes were with Tris-buffered saline containing 5% skim milk and 0.05% Tween-20 for 2 hours at 37°C and then incubated at 4°C overnight with 1:1000 dilutions of the following primary antibodies: anti-β-catenin, anti-phosphorylated β-catenin (p-β-catenin), anti-GSK3β, anti-phosphorylated GSK3β (p-GSK3β), anti-cyclin D1, anti-c-myc, and anti-Runx2. In the case of the anti-GAPDH antibody, a 1:10,000 dilution was used. All of the antibodies were obtained from Cell Signaling Technology, USA. To remove unbound antibodies, the membranes were rinsed three times with Tris-buffered saline for 10 minutes each before being incubated in Tris-buffered saline containing a secondary antibody at a 1:5000 dilution for 1 hour at room temperature. Finally, a chemiluminescence reagent (Thermo, Waltham, MA) was applied to the membranes. The blots were exposed to Kodak X-ray film, and the grayscale values of each protein band were determined using NIH ImageJ 1.34. All of the results were normalized against GAPDH protein levels. The assays were conducted in triplicate.
4
Western Blot Analysis of Hippocampal Proteins
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The hippocampal tissues were homogenized in radioimmunoprecipitation assay lysis buffer supplemented with protease and phosphatase inhibitors. Then, 45 µg protein was resolved by 10% SDS-PAGE and transferred to polyvinylidene difluoride membranes. The blots were blocked with 5% bovine serum albumin for 1 hour before 4°C overnight incubation with appropriate primary antibodies. The blots then were washed with Tris-buffered saline and Tween-20 for three times before incubating with secondary antibodies for 1 hour at room temperature. Finally, chemiluminescence reagent (Thermo Fisher Scientific) was used for detection enhancement and visualization was achieved using Quantity One 1-D Analysis Software (Bio-Rad Laboratories, Inc., Hercules, CA).
5
Western Blot Analysis of Cell Lysates
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Cells were lysed in ice-cold RIPA lysis buffer containing 50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1% Nonidet P-40, 0.5% deoxycholate, 0.1% SDS, 1 mM phenylmethyl sulfonyl fluoride and 20 μg/ml of aprotinin. After centrifugation, cell extracts were resolved by SDS-PAGE and analyzed by immunoblotting. The membranes were probed with the indicated antibodies. Blots were visualized by chemiluminescence reagent (Thermo, Rockford, IL) exposed on X-ray film (Fujifilm). For densitometric quantification, the Western blot images were analyzed with ImageJ 1.48 (Windows version of NIH Image, http://rsb.info.nih.gov/nih-image/ ). The relative amount was calculated and the band with the highest intensity was set as 1. The data was presented as mean values from duplicated experiments.
6
TGF-beta 1 Protein Expression Analysis from FFPE
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Proteins were extracted from formalin-fixed paraffin-embedded (FFPE) tissue using the Qproteome FFPE Tissue Kit (QIAGEN, Mettmann, Germany) according to the manufacturers’ instructions. Extracted proteins were separated using sodium dodecyl sulfate–polyacrylamide gel electrophoresis and blotted on a polyvinylidene difluoride membrane. Membranes were blocked with 5% skim milk powder in Tris-buffered saline [20 mM Tris–HCl and 137 mM NaCl (pH 7.6)] containing 0.1% Tween-20 (TBS-T buffer) for 2 h at room temperature. Membranes were then incubated with antibodies against TGF-beta 1 (Abcam, Cambridge, UK) or β-actin (Cell Signaling Technology, Danvers, MA, USA) overnight at 4 °C. The samples were washed thrice with TBS-T buffer and incubated with the secondary antibodies (Cell Signaling Technology, Danvers, MA, USA) for 1 h at room temperature. After the membranes were washed thrice, protein bands were detected using a chemiluminescence reagent (Thermo Scientific, Waltham, MA, USA). Bands were quantified using ImageJ program (1.43 u; http://rsbweb.nih.gov/ij/ ).
7
Mouse Cytokine Profiling by Ab Array
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To determine cytokines and chemokines in the mouse sera, the Mouse Cytokine Ab Array II kit (AAM-CYT-2-2, RayBio) was used according to the manufacturer's protocol. Pooled serum samples from each group of mice (of days 3 or 7 pi) were applied to the membranes and, after incubation with the detection Ab, membranes were developed with streptavidin-HRP followed by a chemiluminescence reagent (Thermo Scientific). Membranes were then exposed to X-ray film. Pixel densities were calculated for each spot of the array using Quantity One software (Bio-Rad Laboratories) and mean values for duplicate spots were compared. Arbitrary cut-off expression values higher than 1.5-fold were set to consider changes between groups or differences within a group.
8
Immunoblotting Protocol for Protein Analysis
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For immunoblotting, cells were trypsinized and washed with ice-cold PBS, lysed in lysis buffer (50 mM Tris-HCl PH 8.0, 1% SDS, 1 mM EDTA, 5 mM DTT, 10 mM PMSF, 1 mM NaF, 1 mM Na3VO4, and protease inhibitor cocktail), and then denatured in boiling water for 10 min. The cellular lysates were centrifuged (13,000 × g, 30 min), and protein concentration was determined using a BCA assay (ThermoFisher, 23225). Cell lysates were resolved by SDS-PAGE and transferred to polyvinylidene difluoride membranes (Bio-Rad). Membranes were blocked with 5% skim milk and incubated with the indicated antibodies at 4 °C overnight. HRP-conjugated goat secondary antibodies were used (1:5000, Invitrogen). Then membranes were washed three times with TBST followed by secondary antibody incubation for 2 h at room temperature. Immunodetection was achieved with the chemiluminescence reagent (Thermo) and detected by a GE ECL machine.
9
Western Blot Protocol for Protein Analysis
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After harvesting, cells were lysed in an E1A-style cell-lysis buffer [20 mM NaH2PO4, 150 mM NaCl, 50 mM NaF, 0.5% (w/v) IGEPAL, 2.5 mM EDTA, 125 mM sodium pyruvate, and 10% (w/v) glycerol]17 (link), supplemented with a proteinase inhibitor cocktail (1:1000, Millipore Sigma). After 30 min on ice, lysates were centrifuged at 20,000 × g for 10 min at 4 °C to remove cell debris. Protein concentrations in lysates were determined using the Pierce BCA Protein Assay Kit (ThermoFisher Scientific) according to manufacturer’s instructions. Cell lysates were then boiled with sodium dodecyl sulfate (SDS) loading buffer (250 mM Tris–HCl (pH 6.8), 8% (w/v) sodium dodecyl sulfate, 0.2% (w/v) bromophenol blue, 40% (v/v) glycerol, 20% (v/v) β-mercaptoethanol) for 10 min, separated by SDS–polyacrylamide gel electrophoresis (SDS-PAGE) and electroblotted onto nitrocellulose membranes. After blotting, membranes were blocked in 3–5% dry milk (AppliChem) in TBS-T [20 mM Tris, 150 mM NaCl, 0.1% Tween (Millipore Sigma)] and incubated with primary antibodies (Supplementary Table 2 ). After an overnight incubation at 4 °C, membranes were washed three times in TBS-T before 1–2 h incubation with a mouse and/or rabbit secondary antibodies (Supplementary Table 2 ). After the final wash, proteins were detected using a chemiluminescence reagent (Thermo Scientific).
10
Immunoblotting Protein Detection Protocol
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Immunoblotting was performed as described previously [23 (link)]. Briefly, whole-cell lysates were prepared in RIPA lysis buffer containing protease and phosphatase inhibitors, and protein content was determined using a BCA kit (Thermo Fisher Scientific Inc., Rockford, IL, USA). Cellular proteins (30 μg) were separated via SDS-PAGE gel electrophoresis, transferred to a polyvinylidene fluoride membrane, and blotted with specific antibodies at a 1:1000 dilution of antibodies as mentioned in the figure legends. Chemiluminescence signals were obtained using a chemiluminescence reagent (Thermo) and visualized using a ChemiDoc imaging system (Bio-Rad Laboratories, Hercules, CA, USA).
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