Cells were lysed with a 1% Triton X-containing lysis buffer, as described previously [22 (link)]. Lysates were boiled with sodium dodecyl sulfate (SDS) sample buffer for 5 min at 95 °C [22 (link)]. Samples were separated on 7.5, 10, 12 and 15% SDS-PAGE gels and transferred to polyvinylidene difluoride (PVDF) membranes. After blocking with 5% bovine serum albumin (BSA), the membranes were incubated with primary antibodies. The antibodies used were anti-Plectin-1 (#12254), anti-Src pY416 (#2101), anti-Pyk2 (#3292), anti-Pyk2 pY402 (#3291), anti-p38 MAPK (#8690), anti-phospho-p38 MAPK (Thr180/Tyr182) (#9211), anti-p44/42 MAPK (Erk1/2) Antibody (#9102), anti-phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (#4370) (Cell Signaling Technology, CST, Danvers, MA), anti-Src (ab-1) (Merck, Darmstadt, Germany), anti-DDDDK-tag (Flag-tag, Fla-1), anti-GAPDH mAb-horseradish peroxidase-conjugated (HRP)-DirecT (Medical & Biological Laboratories, MBL, Tokyo, Japan), and β-actin (A2228, Sigma Aldrich Chemicals, St. Louis, MO). The membranes were then incubated with HRP-conjugated anti-mouse or anti-rabbit secondary antibodies. Finally, the blots were imaged using a LAS4000 (Fujifilm Wako) with Immobilon ECL Ultra Western HRP Substrate (Merck).
Las 4000
Manufactured by Fujifilm
Sourced in Japan, United States, United Kingdom, Germany, France
The LAS-4000 is a modular imaging system designed for high-resolution and sensitive detection of chemiluminescent, fluorescent, and colorimetric signals. It features a CCD camera, adjustable lighting, and a temperature-controlled imaging chamber to capture detailed images of gels, blots, and other samples.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (39 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (23 mentions)
Ripa buffer, by Thermo Fisher Scientific (22 mentions)
β actin, by Merck Group (19 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (18 mentions)
Protease inhibitor cocktail, by Roche (17 mentions)
Multi gauge software, by Fujifilm (16 mentions)
Nitrocellulose membrane, by Cytiva (16 mentions)
Polyvinylidene difluoride membrane, by Merck Group (13 mentions)
Immobilon western chemiluminescent hrp substrate, by Merck Group (13 mentions)
Bradford assay, by Bio-Rad (12 mentions)
Gapdh, by Cell Signaling Technology (11 mentions)
Pvdf membrane, by Bio-Rad (10 mentions)
Ripa buffer, by Merck Group (9 mentions)
Ecl western blotting substrate, by Thermo Fisher Scientific (9 mentions)
Blocking one, by Nacalai Tesque (9 mentions)
Bradford reagent, by Bio-Rad (9 mentions)
Protease inhibitor cocktail, by Nacalai Tesque (8 mentions)
Enhanced chemiluminescence, by GE Healthcare (8 mentions)
Quick start bradford protein assay, by Bio-Rad (8 mentions)
593 protocols using las 4000
1
Western Blot Protein Analysis Protocol
2
Protein Extraction and Western Blotting
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For protein extraction from GC cell lines, RIPA lysis buffer (Invitrogen; Thermo Fisher Scientific, Inc.) with protease inhibitor cocktail (Sigma-Aldrich; Merck KGaA) was used and then concentration was quantified via Bradford Protein Assay (Bio-Rad Laboratories, Inc.) Protein samples (50 µg/lane) were loaded onto 10% acrylamide gel. Electrophoresis was performed using a Bio-Rad Western Blotting system. (Bio-Rad Laboratories, Inc.) Proteins were transferred to a polyvinylidene fluoride membrane (Sigma-Aldrich; Merck KGaA) and blocked in 5% skimmed milk in Tris-buffered saline (0.1% Tween-20) for 30 min at room temperature. The membranes were incubated with primary antibodies (1:1,000) at 4°C overnight. The antibodies were as follows: Anti-STK31 (cat. no. ab155172; Abcam), anti-Caspase3 (cat. no. 9662; Cell Signaling Technology, Inc.), anti-PARP (cat. no. 9542; Cell Signaling Technology, Inc.) and anti-Tubulin (cat. no. T5168; Sigma-Aldrich; Merck KGaA), then Probed with mouse anti-rabbit IgG conjugated with horseradish peroxidase (1:5,000; cat. no. sc-2357; Santa Cruz Biotechnology, Inc.). Immunopositive bands were visualized using an enhanced luminescence image analyzer LAS-4000 (FUJIFILM Wako Pure Chemical Corporation) and the intensity for each band was estimated by Image J version 1 software (National Institutes of Health).
3
Immunoblotting Protein Quantification Protocol
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For immunoblotting, the cells were washed with PBS and then lysed with a radio immunoprecipitation assay buffer (Bio-Rad Laboratories, Hercules, CA) containing a phospatase inhibitor cocktail 2 (Sigma-Aldrich) and protease inhibitor cocktail (Nacalai Tesque, Kyoto, Japan). The lysates were then centrifuged at 15,000 rpm for 10 min at 4°C. The resultant supernatant was collected, and the protein concentration of the sample was assessed with a BCA™ protein assay kit (Pierce Biotechnology Rockford, IL). The proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride membranes. The membranes were blocked with 5% nonfat dry milk (Cell Signaling Technology, Inc., Danvers, MA) in TBS-T buffer and incubated with the following primary antibodies: Akt, phospho-Akt Ser 473, phospho-paxillin Tyr118, FAK, phospho-FAK Thy397 (Cell Signaling Technology), and GAPDH (Medical & Biological Laboratories CO., Ltd., Nagoya, Japan) for 1 h at room temperature. They were also incubated with HRP-conjugated antirabbit or antirabbit IgG secondary antibody (Cell Signaling Technology) (1:5000 dilutions). The membranes were exposed by an ECL Advance Western blotting detection kit (GE Healthcare, Piscataway, NJ) and then examined with the LAS4000S (Fujifilm, Tokyo, Japan) imaging system.
4
Genotyping rs613872 in TCF4 gene
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Genotyping of rs613872 in TCF4 was performed by PCR using the following primers: forward primer-5′-actgtcaagcactaagcaaagagg-3′, reverse primer- 5′-cccagtagggttgtgatgatgatg-3′. PCR reactions were carried out with Ex Taq DNA polymerase (Takara Bio Inc., Otsu, Japan) under the following conditions: 1 cycle of denaturation at 94°C for 2 minutes followed by 35 cycles of denaturation at 94°C for 30 seconds, annealing at 55°C for 30 seconds, and elongation at 72°C for 30 seconds. Upon completion of the 35 cycles, a final elongation was performed at 72°C for 5 minutes. The PCR products were separated by electrophoresis on 2% agarose gels in Tris-acetate buffer, stained with ethidium bromide, imaged with a luminescence imager (LAS4000S; Fuji Film, Tokyo, Japan), and then extracted from the agarose gels using the a commercial gel and a PCR cleanup system (Wizard SV Gel; Promega, Madison, WI, USA). A terminator cycle sequencing kit and a DNA sequencer (Taq DyeDeoxy and 373A DNA sequencer; Applied Biosystems) were used for direct sequencing.
5
Western Blot Analysis of Cell Signaling
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The MCECs were washed with ice-cold PBS, lysed with ice-cold RIPA buffer containing phosphatase inhibitor cocktail 2 (Sigma-Aldrich Co., St. Louis, MO) and protease inhibitor cocktail (Roche Applied Science, Penzberg, Germany), and then centrifuged. The supernatant representing total proteins was collected and fractionated by SDS-PAGE. The proteins were then transferred to PVDF membranes and blocked with 3% non-fat dry milk, followed by an overnight incubation at 4 °C with the following primary antibodies: phosphorylated focal adhesion kinase (FAK) (1:1000; Cell Signaling Technology, Inc., Danvers, MA), FAK (1:1000; Cell Signaling Technology), phosphorylated MLC (Merck Millipore, Billerica, MA), phosphorylated paxillin (1:1000; Cell Signaling Technology), and GAPDH (1:3000; Abcam, Cambridge, UK). The blots were probed with horseradish peroxidase-conjugated secondary antibodies (1:5000; Cell Signaling Technology), followed by development with luminal for enhanced chemiluminescence using the ECL Advanced Western Blotting Detection Kit (GE Healthcare, Piscataway, NJ), and documentation by an LAS4000S (Fuji Film, Tokyo, Japan) cooled charge-coupled-device camera gel documentation system. Molecular weight markers (Bio-Rad, California) were run alongside all samples. The relative density of the immunoblot bands was determined by Image J® (NIH) software.
6
Western Blot Analysis of EMT Markers
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The iHCECs and iFECDs were washed with ice-cold PBS and then lysed with ice-cold RIPA buffer containing Phosphatase Inhibitor Cocktail 2 and Protease Inhibitor Cocktail. Following centrifugation at 15 000 r.p.m. for 10 min, the supernatant representing total proteins was collected. The protein concentration of the sample was assessed by use of the BCA Protein Assay Kit (Takara Bio, Otsu, Japan). The protein samples were fractionated by SDS-PAGE; proteins were transferred to PVDF membranes. The membranes were blocked with 3% non-fat dry milk for 1 h at RT and were incubated overnight at 4 °C with the following primary antibodies: ZEB1 (1:1000), Snail1 (1:1000), fibronectin (1:1000), and GAPDH (1:3000). After washing, the blots were incubated with horseradish peroxidase-conjugated secondary antibodies (1:5000). The blots were then developed with luminal for enhanced chemiluminescence using the ECL Advanced Western Blotting Detection Kit (GE Healthcare, Piscataway, NJ), documented using an LAS4000S (Fuji Film, Tokyo, Japan).
7
Cell Viability and Proliferation Assay
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HCECs were seeded at a density of 5000 cells/well in a 96-well plate, cultured for 24 hours, and then cultured for a further 24 hours in the presence or absence of inhibitors.
The number of viable cells was evaluated based on the amount of ATP by using a CellTiter-Glo Luminescent Cell Viability Assay using a Veritas Microplate Luminometer (Promega, Fitchburg, WI, USA) according to manufacturer's protocol. Six samples were prepared for each group.
DNA synthesis was detected as incorporation of 5-bromo-2deoxyuridine (BrdU) using the Cell Proliferation Biotrak ELISA system, version 2 (GE Healthcare Life Sciences, Buckinghamshire, UK) according to the manufacturer's instructions. Briefly, HCECs were incubated with 10 lmol/L BrdU for 24 hours, further incubated with fixation solution (Amersham Biosciences, Freiburg, Germany) for 2 hours, and incubated with 100 ll of monoclonal antibody against BrdU for 30 minutes. The BrdU After washing with TBS-T buffer, the blots were incubated with horseradish peroxidase-conjugated secondary antibodies (1:5000: anti-rabbit, anti-mouse IgG; Cell Signaling Technology). The blots were developed with luminal for enhanced chemiluminescence (ECL) using the Chemi-Lumi One Ultra (Nacalai Tesque), documented by LAS4000S (Fuji Film, Tokyo, Japan), and analyzed with Image Gauge software (Fuji Film).
The number of viable cells was evaluated based on the amount of ATP by using a CellTiter-Glo Luminescent Cell Viability Assay using a Veritas Microplate Luminometer (Promega, Fitchburg, WI, USA) according to manufacturer's protocol. Six samples were prepared for each group.
DNA synthesis was detected as incorporation of 5-bromo-2deoxyuridine (BrdU) using the Cell Proliferation Biotrak ELISA system, version 2 (GE Healthcare Life Sciences, Buckinghamshire, UK) according to the manufacturer's instructions. Briefly, HCECs were incubated with 10 lmol/L BrdU for 24 hours, further incubated with fixation solution (Amersham Biosciences, Freiburg, Germany) for 2 hours, and incubated with 100 ll of monoclonal antibody against BrdU for 30 minutes. The BrdU After washing with TBS-T buffer, the blots were incubated with horseradish peroxidase-conjugated secondary antibodies (1:5000: anti-rabbit, anti-mouse IgG; Cell Signaling Technology). The blots were developed with luminal for enhanced chemiluminescence (ECL) using the Chemi-Lumi One Ultra (Nacalai Tesque), documented by LAS4000S (Fuji Film, Tokyo, Japan), and analyzed with Image Gauge software (Fuji Film).
8
Western Blot Detection of JEV-E Protein
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To detect JEV-E protein by western blot analysis, cells or media were harvested. Cells were lysed in lysis buffer (10 mM Tris-HCl, pH 7.4, 10 mM KCl, 1.5 mM MgCl2, 1% (v/v) Triton X-100, 1 mM PMSF, 10 μg/ml leupeptin and 10 μg/ml aprotinin). After incubation on ice for 20 min, debris was removed by centrifugation at 2,000 × g for 10 min at 4 °C. Supernatant and media were used as a loading sample. Proteins (30 μg) were subjected to SDS-poly-acrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes (Millipore, Bedford, MA, USA). Nonspecific binding was blocked by incubation of the membrane with PBS containing 0.1% (v/v) Tween-20 (PBS-T) and 5% (w/v) non-fat dried milk for 20 min at room temperature. Then membrane was incubated with primary antibodies overnight at 4 °C and with secondary antibodies for 45 min at room temperature. Immunoreactive protein bands were visualized using an ECL-peroxidase detection system (Amersham Biosciences, Piscataway, NJ, USA) and LAS-4000 (Fujifilm, Tokyo, Japan), and calculated with Image Gauge. JEV infection levels indicated as the ratio of JEV-E to actin.
9
Western Blotting of Histone, FLAG, and Actin
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Cells were lysed by sonication in ice-cold RIPA buffer containing 1x Protease Inhibitor Cocktail (Roche). Proteins were then separated by SDS-PAGE and transferred to 0.2 µm PVDF membranes (Bio-Rad). Membranes were blocked with Superblock T20 (TBS) blocking buffer (Thermo Fisher Scientific) and probed with primary [anti-Histone H1.2 (Abcam, ab181977, 1:2000 dilution); anti-FLAG M2 (Sigma, F1804, 1:1500 dilution), or anti-beta-actin (Sigma, A2228, 1:5000 dilution)] and secondary [(polyclonal goat anti-rabbit (Dako, P0448, 1:10,000 dilution) or polyclonal goat anti-mouse (Dako, P0447, 1:10,000 dilution)] antibodies or HRP-conjugated p53 antibody (DO1 – sc126, Santa Cruz Biotechnology, 1:1000 dilution). Reactive bands were visualized with Immobilon western chemiluminescent HRP substrate (Millipore) in a luminescence imager (LAS4000, Fujifilm).
10
Protein Expression Analysis of THF-ACG Treated NPC Cells
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Exponentially growth NPC cells were treated with various concentrations of THF-ACGs for the indicated time and cells were lysed by SDS-containing lysis buffer. An equal amount of lysate protein was separated by SDS-PAGE gels, transferred to PVDF membranes and protein expression level was detected by Enhanced chemiluminescence following the manufacturer’s protocol. Images were taken on an LAS-4000 (Fuji Film, Japan).
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