Whole-cell protein lysates were prepared from cell line monolayers according to standard protocols [23 (link)]. Protein concentrations were determined with the Bio-Rad Protein Assay (Bio-Rad Laboratories, Munich, Germany). Proteins were separated by SDS/PAGE as described by Laemmli and colleagues [24 (link)] and transferred to Hybond-P membranes (GE Healthcare, Little Chalfont, UK). Changes in protein expression and phosphorylation as visualized by chemiluminescence (ECL chemi-luminescent reagent, GE Healthcare) were captured and quantified using a FUJI LAS3000 system with Science Lab 2001 ImageGauge 4.0 software (Fujifilm Medical Systems, Stamford, CT).
Ecl chemiluminescent reagent
Manufactured by GE Healthcare
Sourced in United States, Germany, United Kingdom
ECL chemiluminescent reagent is a laboratory product used for the detection and visualization of proteins in Western blot analysis. It is designed to generate a luminescent signal when combined with a specific enzyme-labeled antibody, allowing for the sensitive and quantitative detection of target proteins.
Automatically generated - may contain errors
Lab products found in correlation
Cell lysis buffer, by Cell Signaling Technology (2 mentions)
Image lab software, by Bio-Rad (2 mentions)
Mini protean tgx, by Bio-Rad (2 mentions)
Chemidoc mp imaging system, by Bio-Rad (2 mentions)
Ripa buffer, by Thermo Fisher Scientific (2 mentions)
Hybond p membrane, by GE Healthcare (1 mentions)
Protein assay, by Bio-Rad (1 mentions)
Las 3000 system, by Fujifilm (1 mentions)
Science lab 2001 image gauge 4, by Fujifilm (1 mentions)
Bca assay, by Thermo Fisher Scientific (1 mentions)
Halt proteinase inhibitor, by Thermo Fisher Scientific (1 mentions)
Trans blot turbo system, by Bio-Rad (1 mentions)
Chemidoc touch imaging system, by Bio-Rad (1 mentions)
Laemmli sample buffer, by Santa Cruz Biotechnology (1 mentions)
Anti actin, by Santa Cruz Biotechnology (1 mentions)
Trans blot turbo transfer system, by Bio-Rad (1 mentions)
Non fat dry milk, by GE Healthcare (1 mentions)
Non fat dry milk, by Bio-Rad (1 mentions)
Pard3, by Abcam (1 mentions)
Gapdh, by Abcam (1 mentions)
13 protocols using ecl chemiluminescent reagent
1
Whole-cell Protein Lysate Preparation and Analysis
2
Western Blot Analysis of Transfected HEK293T Cells
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Transfected HEK293T cells were washed with phosphate-buffered saline (PBS) and lysed in radioimmunoprecipitation assay buffer (RIPA, 25 mM Tris (pH 8.0), 137 mM NaCl, 1% glycerol, 0.1% SDS, 0.5% sodium deoxycholate, 1% Nonidet P-40, 2 mM EDTA, and protease inhibitor cocktail) for 20 min on ice. The lysates were clarified by centrifugation (20 min, 14,800 rpm, 4 °C). Samples (cell/viral lysate) were boiled at 95 °C for 5 min with Roti load reducing loading buffer, subjected to SDS-PAGE, and then transferred to a PVDF membrane. Membranes were blocked with skimmed milk solution and probed with appropriate primary antibodies followed by respective secondary antibodies. Signals were visualized using ECL chemiluminescent reagent (GE Healthcare, Munich, Germany).
3
Protein Expression Analysis via Western Blot
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Protein lysates were collected by treating cells with RIPA buffer (Thermo Fisher Scientific) supplemented with HALT Proteinase Inhibitor (Thermo Fisher Scientific) and homogenized via pipetting. Total protein concentrations were calculated via BCA assay (Fisher Scientific); then, 6 μg of each sample was loaded onto a 4–20% Mini-PROTEAN® TGXTM Precast Protein Gel (Biorad) and run at 100 V for 1 h. Samples were transferred to a nitrocellulose membrane by using the Trans-Blot Turbo System (Biorad), blocked in 5% milk, and then incubated with primary antibody (Supplementary Table 1 ) overnight at 4°C and with secondary antibody for 1 h at room temperature. ECL Chemiluminescent Reagent (GE Healthcare Amersham) was applied to the membrane for 5 min, and then the membrane was exposed on the ChemiDoc Touch Imaging System (Biorad). Each marker was examined in at least 4 independent batches of cells.
4
Quantitative Immunoblotting for SCCA1
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Cells were lysed with Cell Lysis Buffer (Cell Signaling Technology, Danvers, MA) supplemented with proteinase/phosphatase inhibitors and PMSF. Equal parts protein (25–30 μg) and Laemmli sample buffer (Santa Cruz Biotechnology, Dallas, TX, USA) were boiled at 95 °C for 10 minutes and gel electrophoresed on 4–20% gradient gels (Mini-Protean TGX, Bio-Rad, Hercules, CA, USA), transferred to PVDF blot using the Trans-Blot Turbo Transfer system (Bio-Rad, Hercules, CA, USA), blocked with 5% milk:TBS-Tween and incubated with 1 : 4000 anti-SCCA1 antibody (NBP2, Novus International, Saint Louis, MO, USA) overnight at four degrees Celsius, 1 : 100 000 anti-Actin (A5441, Santa Cruz Biotechnology, Dallas, TX, USA) or 1 : 2000 anti-GAPDH-HRP conjugated antibody (D16H11, Cell Signaling Technology, Danvers, MA, USA) for 2 h at room temperature. Anti-mouse or anti-rabbit HRP-conjugated secondary antibody was used for detection with ECL chemiluminescent reagent (GE Healthcare Life Sciences, Pittsburgh, PA, USA), visualised, and quantified using the Bio-Rad ChemiDoc MP imaging system and Image Lab software (Bio-Rad, Hercules, CA, USA).
5
Western Blot Analysis of MMP-9 and TIMP-1
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Samples of conditioned media containing 40 µg of total proteins, were mixed with a reducing sample buffer and subjected to 10% SDS-PAGE. Separated proteins were then trans-blotted onto polyvinilidene difluoride (PVDF) transfer membranes. The non-specific protein binding site on membranes was blocked by incubation in a solution containing 5% non-fat dry milk (Biorad, Milan, Italy) in TBS 0.1% Tween 20 (TBS-T) for 1 h, and then incubated overnight at 4 °C with the primary antibodies for human MMP-9 and TIMP-1 diluted in 1% non-fat dry milk in TBS-T. Membranes were then washed in TBS-T, incubated for 1 h with secondary HRP-conjugated antibody diluted in blocking solution, and the immunoreactive bands were detected by inducing a chemiluminescence reaction through the ECL chemiluminescent reagent (GE Healthcare, Little Chalfont, England). Quantitative analysis of immunoreactive spots was performed by using ImageJ software [36 ].
6
Protein Expression Analysis by Western Blot
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Cell lysates were prepared with PLC lysis buffer and were applied onto 10% or 15% SDS-PAGE. After running, the proteins in the gel were transferred onto PVDF membranes. The ECL chemiluminescent reagent (Cat.: RPN2109, GE Healthcare, USA) was used to detect chemical signals. Antibodies including PARD3 (Cat.: ab191204), LC3b (Cat.: ab192890) and GAPDH (Cat.: ab181602) antibodies were purchased from Abcam (Cambridge, MA, USA).
7
Plasma Protein Oxidation Analysis
8
Protein Extraction and Western Blot
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Proteins were extracted using a cell lysis buffer containing 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 5 mM EDTA, protease inhibitors, and 1% Nonidet P-40. A total of 20 μg of protein were separated by SDS-PAGE, electrotransferred onto membranes, and subsequently probed with the indicated antibodies. An ECL chemiluminescent reagent (GE Healthcare Life Sciences, Piscataway, NJ, USA) was used for detection.
9
Investigating Akt Signaling in Cells
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Rabbit monoclonal antibody against pan-Akt and rabbit monoclonal antibody against Akt1 phosphorylated at S473 were purchased from Abcam (Cambridge, UK). Anti-rabbit HRP-conjugated secondary antibody was obtained from Promega (Promega, CO., WI, USA). Collagen was purchased from Invitrogen (Rockville, MD, USA), metformin (1, 1-dimethylbiguanide hydrochloride) (purity > 99%) from Sigma-Aldrich (St. Louis, MO, USA), WST-1 from Roche (Germany), the ECL chemiluminescent reagent from GE Healthcare (Little Chalfont, UK), X-ray films from Agfa HealthCare (Mortsel, Belgium), and the Collagen-based invasion assay from Millipore (Burlington, MA, USA).
10
Western Blot Analysis of p21 and P53
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Cells were lysed with Cell Lysis Buffer (Cell Signaling Technology), proteinase/phosphatase inhibitors and PMSF on ice for 30 minutes and then sonicated using the Bioruptor UCD-200 (Diagenode) on high for 5 minutes. NuPAGE LDS Sample Buffer (4×) (Invitrogen) and SDS buffer (2×) were added, samples were boiled at 95°C for 10 minutes and gel electrophoresed on 4-20% gradient gels (Mini-Protean TGX, Bio-Rad), transferred to PVDF blot, and blocked with 5% TBST milk. Blots were incubated with primary antibodies at 4°C overnight; anti-p21 (1:1000, Cell Signaling Technology [CST], 2947). HRP-conjugated secondary antibodies were incubated 1 hour at room temperature; anti-rabbit (1:4000, sc-2357), P53-HRP (1:1000, sc-126), and actin-HRP (1:5000 sc-47778) (Santa Cruz Biotechnology Inc.) were used with ECL chemiluminescent reagent (GE Healthcare Life Sciences). Blots were visualized and quantified using the Bio-Rad ChemiDoc MP imaging system and Image Lab software (Bio-Rad).
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