Cell extracts were prepared with mammalian protein extraction reagent (M‐PER; Thermo Fisher) plus protease inhibitor cocktail (Halt), and protein concentrations were determined by using the bicinchoninic acid assay (Thermo Fisher). Aliquots of protein lysates were separated on SDS‐10% polyacrylamide gels and transferred to polyvinylidene difluoride membrane filters, which were blocked with 5% blotting grade milk (Bio‐Rad, Hercules, CA, http://www.bio-rad.com ) in TBST (20 mM Tris‐HCl [pH 7.6], 137 mM NaCl, 1% Tween 20). Membranes were then probed with the indicated primary antibodies, reacted with corresponding secondary antibodies, and detected by using a chemiluminescence assay (EMD Millipore, Billerica, MA, http://www.emdmillipore.com ). Membranes were exposed to x‐ray film to visualize the bands (GE Healthcare Life Sciences, Piscataway, NJ, http://www.gelifesciences.com ). The primary antibodies included anti‐p53, anti‐Rb, anti‐Oct4, anti‐SOX‐2, anti‐Nanog, anti‐c‐Myc, anti‐β‐catenin, and anti‐β‐actin (1:1,000; Cell Signaling Technology, Beverly, MA, http://www.cellsignal.com ). The secondary antibodies included horseradish peroxidase‐conjugated donkey anti‐rabbit or anti‐mouse antibodies (1:2,000; GeneTex). The quantification of each protein expression level was normalized by internal control α‐tubulin, and the expression level of parental MSC was referred to as 1.
Chemiluminescence assay
Manufactured by Merck Group
Sourced in United States
Chemiluminescence assay is a laboratory technique that measures the light emitted during a chemical reaction. It is used to detect and quantify analytes, such as proteins, enzymes, and DNA, in a sample. The assay relies on the production of light through a chemical reaction, which can be detected and measured using specialized equipment.
Automatically generated - may contain errors
Lab products found in correlation
Blotting grade milk, by Bio-Rad (5 mentions)
Bicinchoninic acid assay, by Thermo Fisher Scientific (3 mentions)
Protease inhibitor cocktail, by Thermo Fisher Scientific (2 mentions)
Anti c myc, by Cell Signaling Technology (1 mentions)
Anti rb, by Cell Signaling Technology (1 mentions)
Anti sox2, by Cell Signaling Technology (1 mentions)
Anti oct4, by Cell Signaling Technology (1 mentions)
Anti β catenin, by Cell Signaling Technology (1 mentions)
Anti p53, by Cell Signaling Technology (1 mentions)
Anti nanog, by Cell Signaling Technology (1 mentions)
M per, by Thermo Fisher Scientific (1 mentions)
Anti β actin, by Cell Signaling Technology (1 mentions)
Halt protease inhibitor cocktail, by Thermo Fisher Scientific (1 mentions)
Mammalian protein extraction agent, by Thermo Fisher Scientific (1 mentions)
P21 sc 6246, by Santa Cruz Biotechnology (1 mentions)
Gtx133899, by GeneTex (1 mentions)
Gtx107970, by GeneTex (1 mentions)
Phosphatase and proteinase inhibitor, by Beyotime (1 mentions)
X ray film, by Cytiva (1 mentions)
Bca protein assay reagent, by Beyotime (1 mentions)
9 protocols using chemiluminescence assay
1
Western Blot Analysis of Stem Cell Markers
2
Immunoblotting Analysis of EGFR and STAT3
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Cells were lysed and protein was extracted using mammalian protein extraction agent (ThermoFisher Scientific Pierce, Waltham, MA, USA) plus Halt protease inhibitor cocktail (ThermoFisher Scientific Pierce). Protein concentrations were determined using a bicinchoninic acid assay (ThermoFisher Scientific Pierce). Aliquots of protein lysates were separated on sodium dodecyl sulfate‐polyacrylamide gels (SDS‐PAGE) and transferred onto a nitrocellulose membrane, which was blocked with 5% blotting grade milk (BioRad, Hercules, CA, USA) in PBST (0.1% Tween 20 in PBS). The membrane was then hybridized with primary antibodies to human EGFR and phospho‐EGFR (Tyr1068). We also tested the level of STAT3 and phospho‐STAT3 (Tyr705) as STAT3 has been identified to regulate PD‐L1 transcriptionally. Then, with the corresponding secondary antibodies conjugated with horseradish peroxidase, and detected using a chemiluminescence assay (EMD Millipore, Temecula, CA, USA), membranes were exposed to X‐ray film (Kodak China Investment, Shanghai, China) to visualize the bands. β‐actin was used as loading control. All primary and secondary antibodies used in the Western blotting were purchased from Cell Signaling Technology (Danvers, MA, USA).
3
Western Blot Analysis of Cell Signaling
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Cell extracts were prepared with M-PER (Pierce, Rockford, IL, USA) plus protease inhibitor cocktail (Halt™; Pierce, Rockford, IL, USA) and protein concentrations were determined using the BCA assay (Pierce, Rockford, IL, USA) [29 (link)]. Aliquots of protein lysates were separated on SDS-8 or 15% polyacrylamide gels and transferred to PVDF membrane filters, which were blocked with 5% blotting-grade milk (Bio-Rad, Hercules, CA, USA) in TBST (20 mM Tris-HCl (pH 7.6), 137 mM NaCl, 0.1% Tween 20) for 1 h. The filters were then incubated 1 h at room temperature with a 1:1000 dilution in TBST of antibodies against p16 (#92803, Cell Signaling Technology, Danvers, MA, USA), p21(sc-6246, Santa Cruz Biotechnology, Santa Cruz, CA, USA), IKK2 (GTX107970, GeneTex, Hsinchu, Taiwan) and NFκB p65 (phospho Ser536) (GTX133899, GeneTex, Taiwan), reacted with corresponding secondary antibodies, and detected using a chemiluminescence assay (Millipore, Billerica, MA, USA).
4
Western Blot Analysis of Autophagy Markers
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The protein extracts from NPSCs were harvested after lysing with a lysis buffer containing phosphatase and proteinase inhibitors (Beyotime, China). The total protein concentration was determined using the BCA protein assay reagent (Beyotime, China). The protein extracts were subjected to 10% or 12% SDS-PAGE. The protein was separated and then transferred to immunoblot PVD membranes (Millipore, Billerica, MA, USA). The membrane was blocked in 5% blotting-grade milk (Boster Biological Technology Co., Wuhan, China) in TBST for 1 h and then incubated with primary antibodies against P62 (Abcam), LC3 (CST), P27 (Proteintech) and GAPDH (Proteintech) in blocking solution overnight. After washing with TBST for 30 min, the membranes were incubated with the secondary antibody in blocking solution for 2 h at room temperature and then detected using a chemiluminescence assay (Millipore). Finally, the membranes were exposed to X-ray film to visualise the bands (Amersham Pharmacia Biotech). ImageJ was used to quantify the results.
5
Western Blot Analysis of Signaling Proteins
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Cells were lysed in protein extraction reagent (M-PER, Pierce, Rockford, IL) plus protease inhibitor cocktail (Halt™; Pierce). Protein concentrations were determined using the bicinchoninic acid (BCA) assay (Pierce) and heated for 5 min at 95°C in a sample buffer. Equal aliquots of cell lysates were separated on a 10% SDS–polyacrylamide gel. Proteins were transferred to polyvinylidene fluoride (PVDF) membrane filters, blocked for 1 hour, and then probed with specific primary antibodies including pSmad (2/3) (Cell Signaling Technology, Beverly, MA), Smad7 (Santa Cruz Biotechnology, Europe), Sp1 (Santa Cruz Biotechnology, Europe), and β-actin (Novus Biologicals, Littleton, CO). The filter was washed and primary antibodies were allowed to bind by incubating it with horseradish peroxidase-conjugated goat anti-mouse or anti-rabbit IgG (BD Bioscience, San Jose, CA). The filter was washed again; proteins were detected using a chemiluminescence assay (Millipore, Billerica, MA).
6
Evaluating EMT-associated Protein Levels
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To evaluate the expression of EMT associated gene expression in protein levels, we used western blot in the study. Briefly, cells were lysed and protein was extracted using M-PER (Pierce, Rockford, IL, USA) plus protease inhibitor cocktail (Halt; Pierce). Ultrasonic the cell lysis in ice bath (300W for 4 seconds and rest for 8 seconds. Repeat three times.) was Protein concentrations were determined using BCA assay (Pierce). Aliquots of protein lysates were separated on SDS−polyacrylamide gels and transferred onto PVDF membrane, which was blocked with 5% blotting grade milk (Bio-Rad, USA) in PBST (0.1% Tween 20 in PBS). The membrane was then hybridized with the indicated primary antibodies to human E-cadherin, N-cadherin, β-catenin, vimentin, snail, slug and β-actin (Cell Signaling Technology, USA) followed by corresponding secondary antibodies conjugated with horseradish peroxidase, and then detected using a chemiluminescence assay (Millipore, USA). Membranes were exposed to X-ray film to visualize the bands. The band of β-actin was used as endogenous control.
7
Protein Expression Analysis of Pancreatic Tumors in Mice
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Pancreatic tumors in mice were collected and sonicated with lysis buffer, and the protein content was determined using the Bradford assay. An aliquot of the protein solution (10 μg) in each mouse was subjected to sodium dodecyl sulfate-10% polyacrylamide gel electrophoresis (SDS-PAGE), and then electroblotted onto Hybond PVDF membranes (Amersham Bioscience, Little Chalfont, UK). The PVDF membrane was incubated with Tris-buffered saline containing 0.1% Tween 20 and 1 w/v% BSA (1% BSA/TBS-T) at room temperature for 1 h and treated with each primary antibody (diluted 1:1000) in 1% BSA/TBS-T at 4 °C overnight. Subsequently, the membranes were incubated with HRP-conjugated anti-mouse or anti-rabbit secondary antibody-HRP conjugates in 1% BSA/TBS-T at room temperature for 1 h. Protein bands were detected using a chemiluminescence assay (Millipore, Billerica, MA, USA).
8
Western Blot Protein Extraction and Analysis
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Cell extracts were prepared using the M-PER protein extraction reagent (Pierce, Rockford, IL) plus protease inhibitor cocktail (HaltTM; Pierce) and protein concentrations were determined using the bicinchoninic acid (BCA) assay (Pierce). Aliquots of protein 20–40 μg lysates were separated on SDS–10% polyacrylamide gels and transferred to PVDF membrane filters. Membranes were blocked with 5% blotting grade milk (Bio-Rad, Hercules, CA) in TBST (20 mM Tris–HCl [pH 7.6], 137 mM NaCl, 1% Tween 20). Membranes were then probed with the indicated primary antibodies, reacted with the corresponding secondary antibodies, and results detected using a chemiluminescence assay (Millipore, Billerica, MA). Membranes were exposed to X-ray film to visualize the bands (Amersham Pharmacia Biotech).
9
Western Blot Analysis of Ishikawa Cells
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The protein in Ishikawa cells used for western blot was extracted using RIPA lysis buffer (Beyotime Biotechnology, China) supplemented with protease inhibitors (Roche, Switzerland). The proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). After rinsing, the proteins were transferred onto polyvinylidene fluoride (PVDF) membranes and blocked with 5% skim milk, and blotted with the appropriate primary antibodies of P21 (ab219811), Cyclin E1 (ab52189), Cyclin D1 (ab61758), Bid (ab63541), BCL-2-Associated X (Bax, ab32503), active caspase 3 (ab49822), MMP9 (ab38898), MMP2 (ab37150), Sox4 (ab86809), β-catenin (ab6302) GAPDH (ab8245, Abcam, UK) (all dilutions 1:1000) and secondary antibodies. The chemiluminescence assay (Millipore, Germany) was used to detect the antigen-antibody complexes (24 (link)). The relative density was examined using Image Lab™ Software (Bio-Rad, China).
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