For SDS-PAGE and Western blotting (Blair et al., 2009 (link)), the purified protein was first separated by SDS-PAGE on 12% acrylamide gel using Mini-PROTEAN system (Bio-Rad) and transferred to a nitrocellulose membrane (PerkinElmer, Boston, MA, USA) in transfer buffer (25 mM Tris-HCl, 190 mM glycine, 20% methanol, 0.1% SDS, pH 8.3) at 350 mA for 1 h. The membranes were blocked with 10% non-fat dried milk in Tris-bufferred saline with 0.5% Tween-20. After hybridization with anti-His antibodies (Sigma-Aldrich) and peroxidase-conjugated goat anti-mouse secondary antibodies (Millipore, Temecula, CA, USA), the band was visualized with an ECL Western blot kit (PerkinElmer, Boston, MA, USA).
Ecl western blot kit
Manufactured by PerkinElmer
Sourced in United States
The ECL Western blot kit is a laboratory tool used for the detection and analysis of specific proteins within a sample. It provides a chemiluminescent-based method for visualizing and quantifying target proteins separated by gel electrophoresis and transferred to a membrane.
Automatically generated - may contain errors
Lab products found in correlation
Nitrocellulose membrane, by PerkinElmer (1 mentions)
Anti his antibody, by Merck Group (1 mentions)
Mini protean system, by Bio-Rad (1 mentions)
Nitrocellulose membrane, by Merck Group (1 mentions)
Gtx112141, by GeneTex (1 mentions)
Sc 2060, by Santa Cruz Biotechnology (1 mentions)
Sc 2004, by Santa Cruz Biotechnology (1 mentions)
Mab979, by Merck Group (1 mentions)
3 protocols using ecl western blot kit
1
Purified Protein Analysis by SDS-PAGE and Western Blot
2
Quantifying LPS in Bacterial Membrane Vesicles
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To determine the amount of LPS in the BMVs and supernatant, the contents of the BMV preparation and supernatant were separated by polyacrylamide gel electrophoresis and immobilized on nitrocellulose membranes (Millipore, Molsheim, France). The membranes were incubated with primary antibodies, followed by horseradish peroxidase-conjugated goat anti-rabbit (Sigma-Aldrich) secondary antibodies in blocking buffer at room temperature for 4 h. Primary polyclonal antibodies against LPS were generated in rabbits [22 ] and were a gift from Dr. Shih-Hsiung Wu. The ECL Western blot kit (PerkinElmer) was used for chemiluminescent detection. All the BMV samples and supernatant samples used to determine the LPS amount here were in the same volume and thus derived from the same initial bacterial counts.
The production of iNOS in RAW264.7 cells exposed to the prepared BMVs was also determined by western blot analysis. The information about iNOS detection could be found in the supplementary information.
The production of iNOS in RAW264.7 cells exposed to the prepared BMVs was also determined by western blot analysis. The information about iNOS detection could be found in the supplementary information.
3
Western Blot Analysis of EV71 Antigen
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A Western blot analysis was conducted following the procedure used in a previous study [18 (link)]. The abs used to probe EV71 antigen were a mouse anti-EV71 Ab (1:1000; MAB979, Merck Inc., Kenilworth, NJ, USA) as a primary Ab and a horseradish peroxidase (HRP)-conjugated goat anti-mouse polyclonal Ab (1:1000, sc-2060, Santa Cruz Biotechnology, Santa Cruz, CA, USA) as a secondary Ab. The α tubulin was a loading control probed by a rabbit anti-α tubulin primary Ab (1:1000, GTX112141, Gene Tex) and a goat anti-rabbit HRP-conjugated secondary Ab (1:3000, sc-2004, Santa Cruz Biotechnology, Santa Cruz, CA, USA). Proteins were detected using an enhanced chemiluminescence (ECL) Western blot kit (Perkin Elmer Inc., Norwalk, CT, USA).
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