Western-blot of rat hepatocytes developed for beta-actin using Pierce Western blot substrate.
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This product from Thermo Scientific (Pierce) is a cost effective chemiluminescent reagent for detecting antigens immobilized on blots using antibodies conjugated to Horseradish Peroxidase(HRP).
Chemiluminescent detection in Western blotting
Rat primary hepatocyte lysates resolved on SDS-PAGE gel and transferred onto nitrocellulose membrane.
After resolving the protein (50 ug/lane) on a SDS-PAGE (mini-gel), the proteins were transferred onto nitrocellulose membrane. The membrane was blocked with 5% dry milk for 1 hour at room temperature and incubated with 1:10,000 dilution of the ?-actin antibody in 1% dry milk overnight at 4°C. The blot was washed with Tween-Tris-buffered saline (TTBS), 4x, 15 minutes each and then incubated with 1:20,000 dilution of HRP-conjugated secondary antibody (Goat anti-mouse IgG (H + L)-HRP conjugate; Bio-Rad cat #170-6520) for 1 hour at room temperature. The blot was washed 4x with TTBS as before. For chemiluminescent detection, 1 mL of Pierce ECL Western Blotting Substrate Solution A (luminol reagent) was mixed with 1 mL of Solution B (peroxide solution), mixed and overlaid onto the nitrocellulose membrane, which was sitting on a piece of Saran Wrap; the blot was incubated with the detection solutions for 1 minute. The excess reagent was drained by placing the edge of the membrane against a tissue. The membrane was then placed in a clean sheet protector and placed in an film cassette. X-ray film was placed over the membrane and exposed for 1 , 3 and 5 minutes and then developed to visualize the signal.
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A cost-effective regent to detect antigen-antibody complex on Western blots. The reagent works without the need for any optimization, and detects many targets, including housekeeping genes, such as b-actin, and GAPDH. We routinely use this reagent for almost all of our Westerns. If the primary antibody used has good affinity towards its target, this is the go-to chemiluminescent reagent. We have not had any background issues with this reagent. The signal is stable for at least 15 minutes. The bands obtained are quantitative and reproducible.
Make sure the membrane is completely covered with the Pierce ECL reagent during incubation with the reagent.