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Ecl membrane

Manufactured by Cytiva
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The ECL membrane is a type of laboratory equipment used in the process of Western blotting. Its core function is to facilitate the transfer of proteins from a gel to a membrane, which is a crucial step in the analysis and detection of specific proteins within a sample.

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3 protocols using ecl membrane

1

Western Blot Analysis of Extracellular Matrix Proteins

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Following denaturation for 10 min at 65 °C, protein samples were separated by sodium dodecyl sulphate (SDS) 9% polyacrylamide gel electrophoresis (PAGE), and electroblotted onto a Hybond enhanced chemiluminescence (ECL) membrane (Amersham Biosciences). Antibodies for Collagen Type XII, Collagen Type XIV, Collagen Type XI alpha 2, Fibronectin and Prolargin were used. Further details on blocking solutions, primary and secondary antibodies used, as well as their respective host species, working dilutions and commercial suppliers can be found in Supplementary Table 2.
After ECL detection (Amersham Biosciences), bands were quantified using Quantity One 4.6.8 Software (Bio-Rad) and values were normalized to the total protein loading (density value of each complete lane, obtained after staining of the membrane following immunodetection with Page Blue Protein Staining Solution (ThermoFisher Scientific), using a protocol adapted from Welinder and Ekblad28 (link). All samples were run in the same SDS-PAGE gel, and background signal was measured in several different areas of the membrane. Average background signal was then subtracted to the band intensity signal to minimize background variation and interference.
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2

Western Blot Analysis of GSK-3β, pGSK-3β, and β-Catenin

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Immunolabeling of individual GSK-3β and pGSK-3β-ser-9 in cytosol fraction, and β-catenin in cytosol, and nuclear fractions was determined by Western blot. Equal volumes of cytosol and nuclear fractions (20 μl containing 20 μg protein) were resolved onto 7.5% (w/v) SDS-polyacrylamide gel and blotted on enhanced chemiluminescence (ECL) membrane (Amersham, Arlington Heights, IL, USA). Membranes were incubated with polyclonal antibodies for GSK-3β, pGSK-3β-ser-9, and β-catenin overnight at 4°C. The dilution for each antibody was as follows: GSK-3β (1:3000); pGSK-3β-ser-9 (1:1000); and β-catenin (1:1000). The three antibodies were purchased from the Cell Signaling Technology, Inc. (Danvers, MA, USA). They were then exposed to ECL autoradiography film. The same membranes were stripped and reprobed with β-actin (Sigma Chemical Co., St. Louis, MO, USA), which was used as a housekeeping protein. The optical densities (OD) of the bands were quantified using the Loats Image Analysis System (Loats Associates, Inc., Westminster, MD, USA), and the OD of each band was corrected by the OD of the corresponding β-actin band.
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3

Immunoblotting of Rad53 Protein

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Protein extracts were prepared using TCA method (Foiani et al. 1994 (link)). Proteins were separated on 10% acrylamide gels and transferred onto ECL membrane (Amersham). The membrane was blocked with 5% nonfat dry milk in Tris-buffered saline containing 0.05% Tween 20. Proteins were detected using a polyclonal anti-Rad53 antibody (1/10,000, generous gift from JFX. Diffley), and secondary horseradish peroxidase-conjugated antibody (Jackson), and ECL Western blot detection reagents (Amersham).
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