FRT, FRT-ANO1, PC3, Capan-1, and NHNE cells were lysed with cell lysis buffer (50 mM Tris-HCl, pH 7.4, 1% Nonidet P-40, 0.25% sodium deoxycholate, 150 mM NaCl, 1 mM EDTA, 1 mM Na3VO4, and protease inhibitor mixture). Whole cell lysates were centrifuged at 15,000 g for 10 min at 4°C to remove the cell debris, and equal amounts (20, 80 μg protein/lane) of supernatant protein were separated by 4–12% Tris-glycine precast gel (KOMA BIOTECH, Seoul, Korea) and then transferred onto PVDF membrane (Millipore, Billerica, MA). Membrane was blocked with 5% non-fat skim milk in Tris-buffered saline (50 mM Tris-Cl, pH 7.5, 150 mM NaCl) including 0.1% Tween 20 for 1 hour at room temperature. This membrane was then incubated overnight with primary ANO1 antibody (1:500 dilution, ab64085; Abcam Inc., Cambridge, MA). After washing with 0.1% Tween 20 in Tris buffered saline (TBST), the blot was further incubated for 60 min at room temperature with an anti-rabbit secondary antibody (Santa Curz). The membrane was then washed three times with TBST for 3 minutes and then visualized using the ECL Plus western blotting detection system (GE Healthcare Amersham; Piscataway, NJ).
Ecl plus western blotting detection system
Manufactured by GE Healthcare
Sourced in United States, United Kingdom, Japan, Sweden, Spain, Canada, Germany
The ECL Plus Western Blotting Detection System is a chemiluminescent detection system for protein analysis using Western blotting. It provides sensitive and quantitative detection of target proteins from cell and tissue samples.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by GE Healthcare (17 mentions)
Pvdf membrane, by Merck Group (17 mentions)
Amersham hybond p, by GE Healthcare (13 mentions)
Cell lysis buffer, by Cell Signaling Technology (10 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (9 mentions)
Protease inhibitor cocktail, by Roche (8 mentions)
Ripa buffer, by Merck Group (7 mentions)
Pvdf membrane, by Bio-Rad (7 mentions)
Protease inhibitor cocktail, by Merck Group (7 mentions)
Mini protean tgx gel, by Bio-Rad (7 mentions)
Nitrocellulose membrane, by GE Healthcare (6 mentions)
Imagequant las 4000, by GE Healthcare (6 mentions)
Dc protein assay kit, by Bio-Rad (6 mentions)
Nupage 4 12 bis tris gel, by Thermo Fisher Scientific (6 mentions)
Nupage gel, by Thermo Fisher Scientific (6 mentions)
Modified bradford assay, by Bio-Rad (5 mentions)
Mouse anti β actin, by Merck Group (5 mentions)
β actin, by Merck Group (5 mentions)
Polyvinylidene difluoride membrane, by GE Healthcare (5 mentions)
Lds sample buffer, by Thermo Fisher Scientific (5 mentions)
306 protocols using ecl plus western blotting detection system
1
Western Blot Analysis of ANO1 Expression
2
Western Blotting for Protein Quantification
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The Western blotting procedure was performed as described in detail elsewhere for ICDH (33 (link)) and GS (90 (link)). Crude extracts from Prochlorococcus were prepared as described above. Fifteen micrograms of protein was loaded in each lane, subjected to SDS-PAGE, and transferred to a nitrocellulose membrane. After the membrane was blocked, it was incubated overnight with primary antibody (either anti-glutamine synthetase or anti-isocitrate dehydrogenase from Synechocystis sp. PCC 6803, kindly provided by M. I. Muro-Pastor and F. J. Florencio) at the appropriate dilution in Tris-buffered saline with Tween 20 (TBS-T) and 1% bovine serum albumin at 4°C with gentle shaking. The membrane was then washed three times for 15 min each time with TBS-T buffer. The membrane was incubated with a secondary antibody (anti-immunoglobulin from rabbit; labeled with peroxidase; Sigma) diluted 1:2,000 (vol/vol) in TBS-T for 30 min at room temperature with gentle shaking and washed three times for 15 min each time with TBS-T buffer. The immunoreacting material was detected by using the ECL Plus Western blotting detection system (General Electric Healthcare), according to the manufacturer’s instructions. Chemiluminescent signal was detected using a LAS-3000 camera (Fujifilm). Densitometric quantification of the Western blotting bands was performed by using the Quantity One software from Bio-Rad.
3
Western Blot Analysis of MAPK Signaling
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Total proteins of HSC-1C5aR cells were suspended in lysis buffer (7 M urea, 2 M thiourea, 1% Triton X-100, 18 mM dithiothreitol, and 0.1% bromothymol blue) and applied to 12% SDS-polyacrylamide gel electrophoresis gels. The proteins were electrophoretically transferred to an Immobilon Transfer Membrane™ (Millipore, MA, USA) using a semi-dry electroblotter (Sartorius, Göttingen, BRD) for 90 min using a current of 15 V. Phosphorylated and nonphosphorylated anti-p38 MAPK or anti-ERK1/2 rabbit IgGs were obtained from Cell Signaling Technology (MA, USA). Primary antibodies (200 ng/mL) were incubated for 1 h at 22 °C, and HRP-conjugated anti-rabbit IgGs goat IgGs secondary antibody (20 ng/mL; Santa Cruz Biotechnology, CA, USA) was incubated for 30 min at 22 °C. The signals were detected using the ECL-Plus Western Blotting Detection System™ (GE Healthcare, Tokyo, Japan). The phosphorylation ratio of p38 MAPK to ERK1/2 was calculated based on the non-phosphorylated band densities in Western blots using ImageJ 1.46 (National Institutes of Health, MD, USA).
4
Detailed Western Blotting Protocol
5
Protein Expression Analysis by Western Blot
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To analyze the expression of proteins, western blot analysis was performed. Total protein was isolated from the cells using RIPA buffer containing a protease and phosphatase inhibitor cocktail (Thermo Fisher Scientific, Inc.). The lysate was centrifuged at 20,8000 × g for 15 min at 4°C. The supernatants were collected and mixed with 5X loading buffer, followed by incubation in a boiling water bath for 5 min. Protein concentration was determined by Bradford protein assay. Western blot analysis was performed using 12% SDS-PAGE gels for different proteins (50 µg per lane). Following transfer to PVDF membranes, 5% non-fat milk in TBST was used for blocking for 1 h at room temperature, followed by incubation overnight at 4°C with each of the indicated primary antibodies. Subsequently, the membranes were washed with PBST three times for 10 min. The membranes were then probed with HRP-conjugated secondary antibodies (cat. no. ab205719 and cat. no. ab205718; Abcam) in TBST for 1 h at room temperature, and washed with TBST three times. Following washing, enhanced chemiluminescence was used to incubate the membrane and bands were visualized using the ECL Plus Western Blotting Detection system™ (GE Healthcare Life Sciences). Signals were detected and documented with the densitometry system LAS-3000 (Fujifilm).
6
Quantitative Analysis of Kinase Phosphorylation in Intestinal Organoids
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Protein phosphorylation was quantified using the Proteome Profiler Human Phospho-Kinase Array Kit (ARY003, R&D Systems) according to the manufacturer’s instructions. Protein extracts were prepared from untreated hIOs, hIOs in co-culture with human T lymphocytes and hIOs treated with 1 ng/ml rhIL-2. Briefly, hIOs were released from Matrigel using Cell Recovery Solution (Corning) and rinsed with ice cold PBS. hIOs were lysed at 4 °C for 30 min in Lysis Buffer of the Proteome Profiler Human Phospho-Kinase Array Kit (R&D Systems). The phospho-kinase array membranes were blocked, incubated with 200 μg of total protein from hIOs overnight at 4 °C, and then incubated further with cocktails of biotinylated detection antibodies for 2 h at room temperature. Signal was detected with the ECL Plus Western Blotting Detection System (GE Healthcare, Buckinghamshire, UK) and the images obtained underwent quantification by densitometry with Image Gauge software (Fuji Photo Film GMBH, Tokyo, Japan) to determine phospho-protein levels.
7
Western Blot Analysis of Cell Lysates
8
Western Blot Protein Detection Protocol
9
LPS Western Blot Detection
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Western blot analysis of LPS was performed as described previously, using specific antibodies against E. coli LPS (Abcam) (1:5,000 dilution, overnight at 4°C) and donkey anti-rabbit immunoglobulin horseradish peroxidase-linked (GE Healthcare) as a secondary antibody (dilution 1:15,000, 1 h at room temperature) [28 (link)]. The protein-antibody complex was visualized using the ECL Plus Western blotting detection system (GE Healthcare).
10
Western Blot Analysis of Protein Expression
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Proteins were isolated using a modified RIPA lysis buffer containing 50 mM Tris HCl, 1% NP-40, 1% natrium-deoxycholate, 150 mM NaCl, 1 mM EDTA, 1 mM PMSF, 1 mM Na3VO4 and 1x Protease inhibitor cocktail (Roche). Equal amounts of protein were electrophoresed through 4–12% Bis/Tris gels (Life Technologies), transferred to Hybond-P membranes (GE Healthcare Biosciences, Pittsburgh, PA, USA), and immunodetected with rat anti-E2A (clone# 826927, clone R&D Systems, Minneappolis, MN), or rabbit anti-GAPDH (Cat. # G9545, Sigma) antibodies. Bands were detected by chemiluminescence using ECL Plus Western Blotting Detection System and HyperFilm (GE Healthcare Biosciences). Quantification by densitometry was performed using Image J Software [27 (link)].
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