Fresh tissue was lysed in the radioimmunoprecipitation assay buffer (Sigma). Total protein concentrations were detected using a bicinchoninic acid protein assay kit (Beyotime, Shanghai, China). Total protein (20 μg) was separated by 10% SDS-PAGE and transferred to polyvinylidene fluoride membranes (Millipore, Billerica, MA, USA). Then, the membranes were blocked with 10% nonfat milk for 2 h at room temperature. The specific primary antibodies were cultured with the membranes at 4°C overnight. The membranes were incubated with goat anti-rabbit IgG-HRP (1 : 4000; Proteintech) for 2 h at room temperature. Finally, the blots were visualized using the Chemiluminescent Substrate Kit (Thermo Scientific, MA, USA) and the ChemiDoc MP Imaging System (Bio-Rad, CA, USA).
Chemiluminescent substrate kit
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Chemiluminescent Substrate Kit is a laboratory product designed to facilitate the detection and analysis of proteins or other biological molecules through chemiluminescent techniques. The kit provides the necessary reagents and components to perform chemiluminescent assays, which involve the emission of light as a result of a chemical reaction. This product is intended for research and scientific applications.
Automatically generated - may contain errors
Lab products found in correlation
Hrp goat anti rabbit igg, by Proteintech (2 mentions)
Polyvinylidene fluoride membrane, by Merck Group (2 mentions)
Radioimmunoprecipitation assay buffer, by Merck Group (2 mentions)
Chemidoc mp imaging system, by Bio-Rad (2 mentions)
Bicinchoninic acid protein assay kit, by Beyotime (2 mentions)
Polyvinylidene difluoride membrane, by Thermo Fisher Scientific (2 mentions)
Ab6721, by Abcam (2 mentions)
Image pro plus software 6, by Media Cybernetics (2 mentions)
Ab9485, by Abcam (2 mentions)
Bca method, by Thermo Fisher Scientific (2 mentions)
Pvdf membrane, by Bio-Rad (2 mentions)
Protease inhibitor cocktail, by Roche (2 mentions)
X ray film, by Kodak (1 mentions)
Ab238135, by Abcam (1 mentions)
Ab241592, by Abcam (1 mentions)
Tris glycine gel, by Thermo Fisher Scientific (1 mentions)
Phospho nf κb p65 ser536, by Cell Signaling Technology (1 mentions)
Radioimmunoprecipitation assay buffer, by Thermo Fisher Scientific (1 mentions)
Ab19345, by Abcam (1 mentions)
Bicinchoninic acid protein assay kit, by Santa Cruz Biotechnology (1 mentions)
21 protocols using chemiluminescent substrate kit
1
Western Blot Protein Quantification
2
Western Blot Protein Analysis
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Western blot and analysis were carried out, as described previously (Liu et al., 2011 (link)). In brief, proteins (5–10 μg of total protein) were separated by SDS-PAGE, transferred to PVDF membranes, and blocked in 5% skimmed milk. The primary antibodies were rabbit anti-APP (1:2000, ab241592, Abcam), rabbit anti-PSD-95 (1:1000, ab238135, Abcam), or mouse anti-β-actin (1:4000, #3700, CST). The secondary antibodies were corresponding horseradish peroxidase–conjugated ones. A chemiluminescent substrate kit (Thermo Fisher Scientific, United States) was used and developed by using an X-ray film (Kodak) in the dark room. The developed films were scanned, and the percentage of the band relative intensity was analyzed by ImageJ software.
3
Western Blot Analysis of NF-κB Phosphorylation
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Cells were collected in lysis buffer containing protease and phosphatase inhibitors for protein isolation. We prepared cellular extracts by sonication. And total protein concentrations were determined for the Western blot analyses. Proteins were separated on 4%-20% Tris-Glycine gels (Invitrogen, Carlsbad, CA, USA) and transferred to nitrocellulose membranes. After the membranes were blocked in TBST (10 mM Tris-HCl buffer, pH 8.0; 150 mM NaCl; and 0.1% Tween 20) and 5% (w/v) BSA at room temperature for 60 mins, they were incubated overnight at 4 °C with antigen-specific primary antibodies. The primary antibodies used were against phospho-NF-κB p65 (Ser536) (Cell Signaling Technology, Danvers, MA, USA). The blots were under incubation with species-specific HRP-conjugated secondary antibodies for 2 hours at room temperature. And then, the proteins were visualized through incubation with a chemiluminescent substrate kit (Thermo Fisher Scientific, Waltham, MA, USA).
4
Western Blot Analysis of Versican Protein
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Cells were lysed with ice-cold radioimmunoprecipitation assay buffer (Thermo Fisher Scientific, Inc.). The concentration of protein was quantified using a bicinchoninic acid protein assay kit (Santa Cruz Biotechnology, Inc., Dallas, TX, USA). Protein (60 µg/lane) was separated by 12% SDS-PAGE and then transferred onto a polyvinylidene difluoride membrane (Thermo Fisher Scientific, Inc.). The membrane was blocked with 5% non-fat milk (Yili, Beijing, China) in PBS for 3 h at room temperature, and then probed with rabbit anti-human versican antibody (ab19345; 1:50; Abcam, Cambridge, MA, USA) and rabbit anti-human GAPDH antibody (ab9485; 1:50; Abcam) at 4°C overnight. After washing with PBS for 3 times, the membrane was incubated with goat anti-rabbit secondary antibody (ab6721; 1:5,000; Abcam) at room temperature for 40 min. A Chemiluminescent Substrate kit (Thermo Fisher Scientific, Inc.) was used to detect signals according to the manufacturer's protocol. The relative protein expression, represented as the density ratio vs. GAPDH, was determined using Image-Pro plus software 6.0 (Media Cybernetics, Rockville, MD, USA).
5
Detecting CFP-Tagged Protein Expression
6
SALL4 Protein Expression Analysis
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Cells were lysed with ice-cold lysis buffer. Protein was separated with 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then transferred onto a polyvinylidene difluoride membrane (Thermo Fisher Scientific), which was incubated with PBS containing 5% non-fat milk (Yili, Beijing, China) for 3 h at room temperature. After washing with PBS 3 times, the membrane was incubated with rabbit anti-human SALL4 antibody (1:50, ab29112) and rabbit anti-human GAPDH antibody (1:100, ab9485) (both from Abcam, Cambridge, MA, USA) at 4°C overnight. After washing with PBS 3 times, the membrane was incubated with goat anti-rabbit secondary antibody (1:5,000, ab6721; Abcam) at room temperature for 40 min. The Chemiluminescent Substrate kit (Thermo Fisher Scientific) was used to detect signals, according to the manufacturer's instructions. The relative protein expression was analyzed by Image-Pro Plus software 6.0, and presented as the density ratio vs. GAPDH.
7
Protein Extraction and Western Blot Analysis
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Whole tissue or cell proteins were isolated and homogenized by standard methods and analyzed by SDS-PAGE as described previously [40 ]. Protein concentrations were determined by the BCA method (Thermo Scientific). After incubation of membranes with primary antibodies, secondary horseradish-conjugated goat antibodies were added (Supplemental Table S3 ) and a chemiluminescent substrate kit (Thermo Scientific) used for detection. Densitometric analyses were performed by ImageJ [33 (link)].
8
Western Blot Protein Analysis Protocol
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Whole-tissue or whole-cell proteins were isolated and homogenized by standard methods and separated by sodium dodecyl suphate–polyacrylamide gel electrophoresis. Protein concentrations were determined by the BCA method (Thermo Scientific). After incubation with antibodies for RetSat5 (link) (diluted 1:1000 in 4% skimmed milk), ChREBP (NB400-135, lot J1,M1 or M6 (Novus Biologicals, diluted 1:1000 in 0.05% BSA)25 (link) (both antibodies were validated in the referenced studies), Ran (#610340, BD Biosciences, diluted 1:1000 in 4% skimmed milk), Anti-flag (A8592, Sigma, diluted 1:1000 in 4% skimmed milk), β-Actin (sc-47778, Santa Cruz, diluted 1:1000 in 4% skimmed milk), phospho Akt (S473, #4060, Cell Signaling, diluted 1:1000 in 4% BSA), and total Akt (#9272, Cell Signaling, diluted 1:1000 in 4% BSA), secondary horseradish-conjugated antibodies (goat anti-rabbit (#31460, Pierce) and goat anti-mouse (#31440, Pierce), diluted 1:1000-1:5000 in 4% skimmed milk) were added as appropriate and a chemiluminescent substrate kit (Thermo) used for detection.
9
Western Blot Analysis of Protein Levels
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MKN45 cells were treated as described above; after 48 h, the cells were lysed in radioimmunoprecipitation assay buffer (Sigma). Total protein concentrations were detected using a bicinchoninic acid protein assay kit (Beyotime, Shanghai, China). Total protein was separated by 10% SDS–PAGE and transferred to polyvinylidene fluoride membranes (Millipore, Billerica, MA). Then, the membranes were blocked with 8% nonfat milk for 2 h at room temperature. The specific primary antibodies were cultured with the membranes at 4°C overnight. The membranes were incubated with goat anti-rabbit IgG–HRP (1:4000; Proteintech) for 2 h. Finally, the blots were visualized using a Chemiluminescent Substrate kit (Thermo Scientific) and the ChemiDoc MP Imaging System (Bio-Rad).
10
Western Blot Analysis of Protein Targets
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Total protein was subjected to SDS-PAGE and transferred to the PVDF membrane (IPVH00010, Millipore). The antibody against CTSW (ab191083), KLF5 (21017-1-AP), or vinculin (ab219649) was obtained from Abcam or Proteintech. The membrane was incubated with the primary antibody and visualized using the Chemiluminescent Substrate Kit (34580, Thermo Fisher).
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