HaCaT and Hs68 cells were lysed in RIPA buffer containing protease inhibitors and then incubated on ice for 10 min. The protein concentrations of the lysates were normalised by the Bradford method (Bio-Rad Laboratories, Hercules, CA, USA). Equal amounts of protein (20 μg) were resolved by 10% SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes (Whatman GmbH, Dassel, Germany). The membrane was further incubated with specific primary antibodies for 16 h at 4 °C, followed by appropriate secondary antibodies coupled to horseradish peroxidase (Santa Cruz Biotechnology, Santa Cruz, CA, USA) for 2 h. Proteins were developed with ECL Western detection reagents (Amersham Biosciences, Little Chalfont, UK) and visualised by enhanced chemiluminescence (GE Healthcare, Hatfield, UK).
Ecl western detection reagent
Manufactured by Cytiva
Sourced in United States
ECL Western detection reagents are a set of reagents used for the detection and visualization of proteins in Western blot analysis. These reagents enable the chemiluminescent detection of target proteins after they have been separated by gel electrophoresis and transferred to a membrane.
Automatically generated - may contain errors
Lab products found in correlation
Enhanced chemiluminescence, by GE Healthcare (1 mentions)
Horseradish peroxidase, by Santa Cruz Biotechnology (1 mentions)
Bradford method, by Bio-Rad (1 mentions)
Alphaview sa software, by Bio-Techne (1 mentions)
Anti camkii, by Cell Signaling Technology (1 mentions)
Polyvinylidene difluoride membrane, by Merck Group (1 mentions)
Chemiluminescence detection system, by Vilber (1 mentions)
Anti aqp1 antibody, by Abcam (1 mentions)
Horseradish peroxidase conjugated mouse igg, by GeneTex (1 mentions)
Goat anti rabbit secondary antibody, by Cell Signaling Technology (1 mentions)
Iblot dry blotting system, by Thermo Fisher Scientific (1 mentions)
Nupage, by Thermo Fisher Scientific (1 mentions)
Nitrocellulose membrane, by Cytiva (1 mentions)
Total smad3, by Cell Signaling Technology (1 mentions)
P stat3, by Cell Signaling Technology (1 mentions)
Anti p smad3, by Cell Signaling Technology (1 mentions)
Protein assay, by Bio-Rad (1 mentions)
6 protocols using ecl western detection reagent
1
Western Blot Analysis of Protein Expression
2
Western Blot Analysis of CaMK II Expression
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To investigate the expression of putative targets of mmu-miR-152, total protein fractions were purified from allografts and syngrafts as well as normal C3H mice liver. Anti-CaMK II (Cell signaling, USA) and anti-beta-actin (Dawen Biotec, Hangzhou, China) antibody were used for western blot analysis. Equal amounts of protein (40 ug/lane) were resolved by 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred onto a nitrocellulose membrane. After blocking of non-specific binding sites, membranes were incubated overnight at 4°C with Anti-CaMK II antibody (1∶2000), and anti-beta-actin antibody (1∶4000) followed by the corresponding horseradish peroxidase-conjugated secondary antibodies (1∶5000; Dawen Biotec).Then the membranes were developed in the ECL Western detection reagents (Amersham–Pharmacia Biotech, Piscataway, NJ, USA), according to the manufacturer's protocol. The gray value of each band was analyzed and auto-calculated by AlphaView SA software (Alpha Innotech, USA).
3
Protein Expression Analysis after UVB Exposure
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Cells were harvested after UVB treatment for 24 and 48 hours, washed thrice with PBS and resuspended in ice-cold buffer containing 50 mM Tris, 150 mM NaCl, 1 mM ethylenediaminetetraacetic acid, 1% Triton X-100, 0.5% sodium deoxycholate, and protease/phosphatase inhibitors (GenDEPOT, Barker, TX, USA). Cell lysates were centrifuged at 12,000 g for 10 minutes, and the supernatants were collected. Samples were separated on 6.5% to 12% sodium dodecyl sulfate-polyacrylamide gels and transferred to polyvinylidene difluoride membranes (Millipore, Billerica, MA, USA). Membranes were blocked with 5% skimmed milk prepared in Tris-buffered saline containing 0.1% Tween-20 and incubated with an anti-AQP1 antibody diluted 1:500 (Abcam) overnight at 4oC. After that, membranes were washed and incubated with horseradish peroxidase-conjugated mouse IgG diluted 1:2,500 (GeneTex, Irvine, CA, USA) at room temperature for 1 hour. Complexes were visualized by enhanced chemiluminescence using ECL western Detection Reagents (Amersham Pharmacia Biotech, Piscataway, NJ, USA). Signals were detected using a chemiluminescence detection system (Vilber, Collégien, France).
4
Western Blot Analysis of BV2 Microglia Proteins
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BV2 microglia were harvested and lysed in RIPA buffer [1 mM ethylenediaminetetraacetic acid (EDTA), 150 mM NaCl, 1% igepal (CA-630), 0.1% sodium dodecyl sulfate (SDS), 0.5 % sodium deoxycholate and 50 mM Tris HCl; pH 8.0 (Sigma)]. Equal amounts of protein were separated by 8–12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), transferred to polyvinylidene fluoride (PVDF) membranes, blocked with 5% nonfat milk for 2 h and incubated with primary antibodies at 4°C overnight. Following incubation with appropriate secondary antibodies conjugated to horseradish peroxidase (goat anti-rabbit secondary antibody was obtained from Cell Signaling Technology), immunoblots were exposed on film using electrochemiluminescence (ECL) western detection reagent (Amersham Pharmacia Biotech, Amersham, UK). The bands were quantified by the optical density ratio using β-actin as a control.
5
Western Blot Analysis of Cellular Proteins
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Proteins were loaded on NuPAGE™ 3-8% Tris-Acetate (for visualizing MCM-7, MRCK and MYPT) Protein Gels or NuPAGE 4-12% Bis-Tris (all other proteins) Protein Gels (Invitrogen) and run at 150V for 90 minutes, after which time they were transferred to nitrocellulose membranes using an iBlot dry blotting system (Invitrogen). Blots were then washed 3x 5 minutes in PBS, following by blocking in 5% milk in PBS-T (PBS plus 0,1% Tween). Primary antibodies were then incubated in 5% BSA in PBS-T overnight at 4°C. Blots were washed 3x 5 minutes in PBS-T, following by incubation with secondary antibodies for 1 hour in 5% milk in PBS-T. Blots were washed 3x 5 minutes in PBS-T and proteins were visualized using ECL western detection reagent (Amersham) and imaged with X-ray films.
6
Western Blot Analysis of Cellular Proteins
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