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Amersham ecl western blotting substrate

Manufactured by GE Healthcare
Sourced in United Kingdom

The Amersham ECL Western blotting substrate is a chemiluminescent detection system used for the visualization of proteins in Western blot analysis. It generates a measurable light signal in proportion to the amount of target protein present in the sample.

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5 protocols using amersham ecl western blotting substrate

1

Western Blot Analysis of EMT Markers

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Protein lysates were prepared from cells using 1X RIPA lysis buffer (Merck) and western blot analysis was performed as described 7 (link). The membranes were probed with primary antibodies overnight at 4 °C. Antibodies against SMAD4 (at 1:500 dilution) (cat. no. 3854), SNAI1 (1:250) (cat. no. 3879), E-cadherin (CDH1) (1:1,000) (cat. no. 3195) and GAPDH (1:2,000) (cat. no. 5174) were obtained from Cell Signaling Technology, MA, USA. The proteins were detected using a horseradish peroxidase-conjugated secondary antibody (1: 5,000, Abcam, Cambridge, UK) (cat. no. ab205718) incubating for 1 h at room temperature. Protein bands were visualized with the Amersham ECL western blotting substrate (GE Healthcare, PA, USA), according to the manufacturer's protocol.
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2

Western Blot Analysis of Epithelial-Mesenchymal Transition

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Protein lysates were harvested from the cells using 1× RIPA lysis buffer (Nacalai Tesque, Kyoto, Japan) and kept on ice for 30 min before being centrifuged at 10,000 g for 10 min at 4 °C to obtain the supernatant. Protein lysates (50 μg) were subjected to SDS-polyacrylamide gel electrophoresis and western blot analysis as described [19 (link)]. Dilutions and the sources of the antibodies used are as follows: E-cadherin (CDH1, 1:1000, Cell Signaling Technology), occludin (OCLN, 1:1000, Merck Millipore), vimentin (VIM, 1:1000, Cell Signaling), SNAI1 (1:250, Cell Signaling) and GAPDH (1:2000, Cell Signaling). The protein bands were detected using a horseradish peroxidase-conjugated secondary antibody (1: 10,000, Abcam, Cambridge, UK) for 1 h at room temperature and visualized with the Amersham ECL Western blotting substrate (GE Healthcare), according to the manufacturer’s protocol. The mean of multiple blots is presented (Fig. 4b); the error bars represent standard errors of the mean, SEM.
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3

Western Blot Analysis of Protein Expression

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NPDFs were lysed using PRO-PREPTM protein extraction solution (iNtRON Biotechnology, Seongnam, Korea). Cell lysates were subjected to SDS-PAGE and transferred to polyvinylidene difluoride membranes (Millipore Inc., Billerica, MA). Membranes were probed with primary antibodies overnight at 4 °C. The primary antibodies used included the followings: anti-α-SMA (Chemicon, Millipore Inc., Billerica, MA), anti-acetyl H3 (Cell Signaling), anti-histone H3, anti-fibronectin, anti-phospho-smad2/3, anti-total smad2/3, and glyceraldehyde-3-phosphate dehydrogenase (Santa Cruz Biotechnology, Inc., Santa Cruz, CA). Membranes were washed for 5 minutes three times, incubated with peroxidase-conjugated anti-mouse or anti-rabbit antibodies (Vector Laboratories, Burlingame, CA) for 1 hour, and washed for 5 minutes three times. Protein expression was detected using Amersham ECL Western Blotting Substrate (GE Healthcare, Little Chalfont, Buckinghamshire, UK).
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4

Western Blot Analysis of Nuclear Proteins

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Nuclear extracts were obtained using a lysis buffer (M-PER protein extraction reagent for the cells, T-PER protein extraction reagent for the tissues) supplemented with a 1:100 protease inhibitor cocktail (Pierce). The protein concentrations were measured using a Bradford Protein Assay kit (Bio-Rad) according to the manufacturer’s instructions. The proteins (10 mg per sample) were resolved on an SDS-PAGE gel and transferred to nitrocellulose membranes (GE Healthcare). The membranes were blotted with specific antibodies (SMAD2, α-SMA, FN, p-SMAD2 or GAPDH) overnight at 4 °C. The membranes were subsequently washed with TBST for 30 minutes at room temperature, incubated with horseradish peroxidase-conjugated secondary antibodies for 60 minutes, and washed a second time with TBST for 30 minutes. Protein expression was detected using Amersham ECL Western Blotting Substrate (GE Healthcare).
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5

Western Blot Analysis of Cellular and Viral Proteins

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HAP1 cells were trypsinized and collected by centrifugation at 350 rcf for 10 min. The pellet was resuspended in Laemmli sample buffer containing 100 mM DTT, boiled for 5 min at 95°C and subjected to electrophoresis in 10% acrylamide (Bio-Rad) gels. Viruses were purified and concentrated over a 20% sucrose cushion (in TN buffer) at 110,000 rcf. Pelleted virus was resuspended in TN buffer overnight on ice. After addition of Laemmli sample buffer (1× final concentration, 100 mM DTT), samples were boiled for 5 min at 95°C and subjected to electrophoresis in 7% acrylamide (Bio-Rad) gels. Upon transfer to a nitrocellulose membrane (Millipore), the presence of cellular and viral proteins was probed with antibodies against GM130 (rabbit pAb, Abcam), FLAG (HRP-labeled mouse anti-FLAG mAb, Sigma) or the S2 subunit of MHV A59 [105] (link) (mouse anti-S2 mAb) diluted 1∶1000. When necessary, the blots were subsequently incubated with HRP-labeled rabbit anti-mouse or swine anti-rabbit antibodies (both diluted 1∶5000; DAKO). Binding of HRP-labeled antibodies was visualized using Amersham ECL Western blotting substrate (GE Healthcare Life Sciences) according to the manufacturer's instructions.
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