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.
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.
Automatically generated - may contain errors
Lab products found in correlation
Horseradish peroxidase conjugated secondary antibody, by Abcam (2 mentions)
Acrylamide, by Bio-Rad (2 mentions)
Ripa lysis buffer, by Merck Group (1 mentions)
Snai1, by Cell Signaling Technology (1 mentions)
E cadherin cdh1, by Cell Signaling Technology (1 mentions)
Vimentin vim, by Cell Signaling Technology (1 mentions)
Gapdh, by Cell Signaling Technology (1 mentions)
Polyvinylidene difluoride membrane, by Merck Group (1 mentions)
Anti fibronectin, by Santa Cruz Biotechnology (1 mentions)
Anti phospho smad 2 3, by Santa Cruz Biotechnology (1 mentions)
Glyceraldehyde 3 phosphate dehydrogenase, by Santa Cruz Biotechnology (1 mentions)
Anti acetyl h3, by Cell Signaling Technology (1 mentions)
Anti α sma, by Merck Group (1 mentions)
Anti histone h3, by Cell Signaling Technology (1 mentions)
Pro prep protein extraction solution, by iNtRON Biotechnology (1 mentions)
Nitrocellulose membrane, by GE Healthcare (1 mentions)
Bradford protein assay kit, by Bio-Rad (1 mentions)
Nitrocellulose membrane, by Merck Group (1 mentions)
Rabbit pab, by Abcam (1 mentions)
5 protocols using amersham ecl western blotting substrate
1
Western Blot Analysis of EMT Markers
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.
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).
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).
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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