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Fluorescent detection with Blocker FL vs competitors
Western blotting was performed by loading serial dilutions of A431 cell lysate (15 mg, 3-fold serially diluted) and carrying out electrophoresis on a Novex 4-20% Tris-Glycine Midi Plus gel (Cat. No. WXP42012BOX). The last 2 lanes were loaded with 5 µL PageRuler Prestained Protein Ladder (Cat. No. 26616) and 3 µL iBright Prestained Protein Ladder (Cat. No. LC5615). The proteins were transferred to a nitrocellulose membrane using the Pierce Power Blotter (Cat. No. 22834). The membranes were blocked for 30 minutes with either 1X Thermo Scientific Blocker FL Fluorescent Blocking Buffer (Cat. No. 37565), Odyssey Blocker in TBS, or a homebrew of 5% non-fat milk in 0.05% Tween TBS.
The membranes were probed with the following primary antibodies diluted in the corresponding blocking buffers: Chicken anti-calreticulin, 1 mg/mL (Cat. No. PA1-903); Rabbit anti-EGFR, 1 mg/mL (Cat. No. 700308); Mouse anti-stat3, 0.5 mg/mL (Cat. No. MA1-13042); Mouse anti-ras10, 0.5 mg/mL (Cat. No. MA1-012). The membranes were washed and probed with the following secondary antibodies in TBS-Tween: Goat anti-chicken Alexa Fluor 546, 0.2 mg/mL (Cat. No. A11040); Goat anti-rabbit Alexa Fluor Plus 800, 0.2 mg/mL (Cat. No. A32735); Goat anti-mouse Alexa Fluor Plus 680, 0.2 mg/mL (Cat. No. A32729). Membranes were washed and imaged using the iBright FL Imager (Cat. No. A32752).
