Equal amounts of protein were separated by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to PVDF membranes (Sigma-Aldrich, St. Louis, MO, USA) with Mini-PROTEAN® Tetra Handcast Systems (Bio-Rad, Hercules, CA, USA). The membranes were incubated with primary antibodies (for anti-GFP (Abcam, AB6556), 1:5000; anti-HA (Roche, 11867423001), 1:5000; anti-PR1, 1:5000; anti-RbCL (Clontech 632475), 1:2000; anti-GST (Virogen, 101-A-100), 1:5000; or anti-His (Thermo Fisher, PA1-23024), 1:5000) in 1× TBS with 5% w/v skim milk at 4 °C overnight. Treated membranes were washed with 1× TBS and incubated with a secondary horse radish peroxidase (HRP)-conjugated antibody (anti-rabbit HRP (Santa Cruz, sc-2004), 1:10,000; anti-rat (Santa Cruz, sc-2006), 1:10,000; anti-mouse (GE Healthcare Life Science; NA931), 1:10,000) at room temperature for 2 h. Signals were visualized using the Pierce ECL and ChemiDoc MP (Bio-Rad) systems. Protein band intensity was measured by using ImageJ software. Relative intensity protein band under Pto infection condition was calculated by normalizing to the intensity of protein band under mock condition, which was set as 1.
Pierce ecl
Manufactured by Bio-Rad
The Pierce ECL is a chemiluminescent Western blotting detection reagent. It is designed to detect and visualize proteins on membranes after electrophoretic transfer and immunoblotting.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (2 mentions)
Mini protean tetra handcast system, by Bio-Rad (1 mentions)
Chemidoc mp, by Bio-Rad (1 mentions)
Na931, by GE Healthcare (1 mentions)
Anti ha, by Roche (1 mentions)
Clarity ecl, by Bio-Rad (1 mentions)
Nupage gel, by Thermo Fisher Scientific (1 mentions)
Chemidoc imaging system, by Bio-Rad (1 mentions)
X ray film, by Kodak (1 mentions)
Hrp conjugated secondary antibody, by Cell Signaling Technology (1 mentions)
2 protocols using pierce ecl
1
Quantitative Western Blot Analysis of Pathogen-Induced Proteins
2
Western Blot Protein Analysis Protocol
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Extracts were analyzed by SDS–PAGE using a 4–12% Bis–Tris or 3–8% Tris‐Acetate NuPAGE gels (Invitrogen) and transferred onto PVDF membranes (Millipore). After being washed with 1× PBS containing 1% Tween‐20 (PBS‐T), the membranes were blocked in 5% milk or 5% bovine serum albumin (BSA) in PBS‐T for 1 h at RT and then incubated with the primary antibodies overnight at 4°C. The membranes were incubated with HRP‐conjugated secondary antibodies (Cell Signaling) for 1 h at RT and exposed to X‐ray film (Kodak) or the ChemiDoc Imaging system (Biorad) after incubation with Thermo Scientific Pierce ECL or Clarity ECL (Biorad).
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